Introduction

The genus Rumex with more than 200 species distributing widely in the world is the second largest genus in the family Polygonaceae, in which some species displayed nutritional and medicinal properties. For example, Rumex patientia L. was reported having abundant amino acid and cellulose [1]. A series of anthranoids, tannins, naphthalenes, and flavonoids were identified as the major chemical compositions from Rumex species [2,3,4,5].

Rumex crispus L., a perennial herbaceous species with stout and straight root system, is widely distributed in China, Korea, Kazakhstan, Russia, Japan, Europe, and North America [6, 7]. It has been used medicinally for treating jaundice and related liver diseases, stomachache, neckache, low blood pressure, pneumonia, wound healing, and rheumatism [8,9,10]. The crude extract was reported to possess anti-inflammatory, antimicrobial, antioxidant, and anti-diabetic properties [11,12,13,14]. However, the chemical compositions are so far not well-known. Our detailed phytochemical study on the roots of R. crispus led to the isolation of four new compounds including a seco-anthraquinone glucoside (1) and three naphtholones (2–4), along with 10 known anthraquinones (6–14) and napthalenone (5) (Fig. 1). Most of the isolates, 1 and 614, were evaluated for their anti-fungal activity against three skin fungi (Epidermophyton floccosum, Trichophyton rubrum, Microsporum gypseum), and the anti-inflammatory properties. Herein, we report the study.

Fig. 1
figure 1

Compounds 114 isolated from the roots of Rumex crispus L.

Result and discussion

The air-dried roots of R. crispus were crushed into small grains and extracted with 90% aqueous MeOH. After removal of the organic solvent, the crude extract was suspended into water and fractionated with ethyl acetate. The ethyl acetate fraction was further applied to repeated column chromatography (CC) over Sephadex LH-20, macroporous resin D101, silica gel, and RP-18, followed with semipreparative HPLC, yielded 14 compounds. Among which, one seco-anthraquinone glucoside (1) and three naphtholones (24) are new compounds. Ten known compounds were identified as 3-acetyl-2-methyl-1,4,5-trihydroxy-2,3-epoxynaphthoquinol (5) [15], nepalensides A (6) and B (7) [16], polyanthraquinoside A (8) [17], emodin (9) [18], physcion (10) [19], chrysophanol (11) [20], emodin-1-O-β-D-glucopyranoside (12) [21], 6-methoxyl-10-hydroxyaloin B (13) [22], (10R)-3-methyl-1,8,10-trihydroxy-10-D-glucopyranosyl-9(10H)-anthracenone (14) [23] (Fig. 1), respectively, by comparison of their spectroscopic data with literature values. Seven compounds, 58 and 1214, were isolated from R. crispus for the first time.

Structural identification of compounds

Compound 1 was obtained as yellowish amorphous powder. Its molecular formula, C21H22O11, was determined by negative HRESI-TOF–MS (m/z 449.1094 [M–H], calcd. for C21H21O11, 449.1089). In the 13C NMR spectrum of 1 (Table 1), 14 carbon signals due to one ketone (δC 203.3), one carboxyl (δC 170.8) and two benzene ring (δC 106.0–165.0, 12 × C) were observed, assignable to a carboxylated benzophenone. In addition, 6 carbon signals at δC 101.3 (C-1″), 74.7 (C-2″), 78.2 (C-3″), 71.7 (C-4″), 77.8 (C-5″), and 62.5 (C-6″) from a glucosyl moiety and a methyl signal at δC 21.4 were also observed. In the 1H NMR spectrum of 1, three characteristic proton resonances at δH 6.61 (1H, d, J = 8.3 Hz, H-4), 7.36 (1H, t, J = 8.3 Hz, H-5) and 6.56 (1H, d, J = 8.3 Hz, H-6) suggested the existence of a 1,2,3-trisubstituted benzene ring [24]. Moreover, two aromatic singlet resonances at δH 6.90 (1H, s, H-3′) and 7.34 (1H, br s, H-5′)] and a 3-H singlet signal at δH 2.38 (3H, s) due to a methyl group were observed, together with one anomeric proton at δH 4.85 (1H, d, J = 7.8, H-1″) and a set of proton signals in the range between δH 2.3–3.8, disclosing the existence of a sugar moiety. The J1″−2″ coupling constant of anomeric proton (7.8 Hz) revealed the glucosyl anomeric center to be β configuration. The 1H and 13C NMR data of 1 were closely related to those of 6 [16]. However, instead of a symmetric 1,2,3-trisubstituted benzene ring in 6, an un-symmetric 1,2,3-trisubstituted benzene ring appeared in 1. This was confirmed by the HMBC correlations of H-4 (δH 6.61) with C-1 (δC 114.9), C-2 (δC 165.0), C-3 (δC 160.0) and C-6 (δC 106.0), and H-6 (δH 6.56) with C-1 (δC 114.9), C-4 (δC 112.4) and C-7 (δC 203.3). Moreover, the HMBC correlations from methyl proton at δH 2.38 to C-3′ (δC 121.8), C-4′ (δC 140.8), and C-5′ (δC 122.8), from H-3′ (δH 6.90) to C-5′ and C-6′ (δC 132.3), and from H-5′ (δH 7.34) to C-3′, C-6′ and C-7′ (δC 170.8) (Fig. 2) confirmed the structure of 1. Therefore, the structure of compound 1 was established as shown in Fig. 1 and named as crispuside A.

Table 1 1H (600 MHz) and.13C (150 MHz) NMR data of 1 in CD3OD (δ in ppm, J in Hz)
Fig. 2
figure 2

Key 1H–1H COSY and HMBC correlations of 1, 2, 3 and 4

Compound 1 is a new anthraquinone-related compound, whose formation mechanism might be similar to that of desmethylsulochrin, which was established by the ring-opening process of questin catalyzed by GedF (Geodin synthesis protein F) and GedK [25]. In R. crispus, compound 1 maybe formed from reduction firstly and then ring-opening of ziganein-1-O-β-glucopyranoside catalyzed by GedF and GedK respectively (Fig. 3).

Fig. 3
figure 3

Possible formation of compound 1

Compounds 2 and 3, obtained as colorless powder, are a pair of enantiomers possessing the same molecular formula C13H16O4, as deduced by the negative HRESIMS (m/z 235.0979 [M−H] calcd C13H15O4, 235.0976). The 1H, 13C NMR, and HSQC spectral data of 2 and 3 revealed the existence of two methyls [δH 2.48 (3H, s, H-11), δC 21.1; δH 1.52 (3H, d, J = 6.7 Hz, H-10), δC 21.1], two methylenes [δH 2.88 (1H, m, H-2a), 2.67 (1H, m, H-2b), δC 35.9; δH 2.27 (1H, m, H-3a), 2.08 (1H, m, H-3b), δC 32.6], one aromatic methine [δH 6.89 (1H, s, H-5), δC 121.9], two oxymethines [δH (1H, 4.78, dd, J = 8.0, 3.8 Hz, H-4), δC 68.1; δH 5.34 (1H, q, J = 6.7 Hz, H-9), δC 65.6], one carbonyl group (δC 206.1, C-1), and a set of quaternary aromatic carbons (δC 146.4, 147.2, 130.9, 161.5, 114.6). These spectroscopic features were similar to those of (4S,9S)-9-hydroxy-O-methylasparvenone (4S,9S-HM) [26], whose molecular weight was 252 Da, 16 Da more than those of 2 and 3. However, the chemical shifts of C-6 and C-11 in 2 and 3 were obviously different with those of 4S,9S-HM, indicating that the substituent at C-6 in 2 and 3 was methyl group, instead of a methoxyl group in 4S,9S-HM. Moreover, the HMBC correlations of H-11 (δH 2.48) with C-5 (δC 121.9), C-6 (δC 147.2) and C-7 (δC 130.9) confirmed the substitution of methyl at C-6 position. The 1H–1H COSY correlations between H-2 and H-3, and the key HMBC correlations from H-2 to C-1/C-3, from H-3 to C-1/C-2, from H-4 to C-5/C-8a, from H-11 to C-5/C-6/C-7, from H-5 to C-8a, from H-10 to C-7/C-9, from H-9 to C-6/C-7/C-8 determined the planar structures of 2 and 3 as 4,8-dihydroxy-7-(1-hydroxyethyl)-6-methyl-3,4-dihydronaphthalen-1(2H)-one. Comparison of the experimental ECD spectrum of 2 with the calculated ECD (Fig. 4) of the four stereoisomers, (4S,9S)-, (4R,9R)-, (4S,9R)- and (4R,9S)- HM [26] showed the ECD of 2 was more comparable with the computationally derived data for (4S,9S)- and (4S,9R)- with positive Cotton effects (CEs) at 248 and 214 nm and negative CE at 280 nm. However, the CE amplitudes of 2 were notably closer to those of the (4S,9S)- diastereomer, thus favoring the (4S,9S) absolute configuration for 2. Therefore, compound 2 was proposed as (4S,9S)-4,8-dihydroxy-7-(1-hydroxyethyl)-6-methyl-3,4-dihydronaphthalen-1(2H)-one. Similarly, the absolute configuration of 3 was deduced as (4R,9R)-4,8-dihydroxy-7-(1-hydroxyethyl)-6-methyl-3,4-dihydro-naphthalen-1(2H)-one. The structures of 2 and 3 were established as shown and named as naphthalenones A (2) and B (3), respectively.

Fig. 4
figure 4

Experimental ECD curves of 2, 3, 4 and ADMT, and calculated ECD curves of four stereoisomers of HM

Compound 4 was isolated as white powder. Its molecular formula was assigned to be C13H14O4, as deduced from the HRESIMS (m/z 233.0814 [M−H], calcd. C13H13O4, 233.0814). The UV spectrum showed absorption bands at λmax 286 nm. The 1D and 2D NMR data of 4 (Table 2) indicated it shared the same 3,4-dihydronaphthalen-1(2H)-one skeleton with 2. The molecular weight of 4 is 2 Da less than that of 2. Comparing the DEPT and 13C NMR data of 4 with those of 2 indicated that, a ketone group (δC 206.5) appeared in 4, instead of an oxymethine C-9 (δC 65.6) in 2. This was further verified by the HMBC correlation of H-10 with C-9 in the HMBC spectrum of 4. Comparing with the ECD curve of the known 7-acetyl-4R,8-dihydroxy-6-methyl-1-tetralone (ADMT) [27], compound 4 showed a negative CE at 260–270 nm in ECD spectrum, which is in contrast to ADMT (Fig. 4). Therefore, the structure of 4 was determined to be 7-acetyl-4S,8-dihydroxy-6-methyl-1-tetralone and named as naphthalenone C.

Table 2 1H (600 MHz) and13C (150 MHz) NMR data of 2, 3, 4 and 5 in CD3OD (δ in ppm, J in Hz)

Compound 5 was isolated as colorless needle crystal with a molecular formula of C13H14O5, as determined by the ESI–MS (negative ion mode) m/z 249 [M−H], and 13C NMR and DEPT spectroscopic data. The 13C NMR spectrum of 5 showed one carbonyl (δC 208.6), six aromatic carbon signals (δC 157.4, 116.0, 129.9, 119.1, 137.8, 120.2) with three methines and one oxygenated quarternary carbon, and six sp3 carbon signals (δC 70.2, 65.6, 72.6, 68.1, 29.1, 15.9) assignable to two methyls, two oxymethines and two oxy quarternary carbons. The 1H NMR spectrum showed three characteristic proton resonances at δH 6.72 (1H, d, J = 7.9 Hz, H-6), 7.17 (1H, t, J = 7.9 Hz, H-7), 7.10 (1H, d, J = 7.9 Hz, H-8) suggesting the existence of a 5,9,10-trisubstituted benzene ring. In addition, two methyls (δH 2.36, s, H-12; 1.47, s, CH3-2) and two oxymethines (6.72, d, J = 7.9 Hz, H-6; 5.57, s, H-4) signals were observed. The above data indicated that 5 was a naphthoquinol derivative, whose epoxide ring was inferred by chemical shifts (δC 65.6, C-2; δC 72.6, C-3) and seven degrees of unsaturation. The detailed analyses of the 2D NMR (1H-1H COSY, HMQC, HMBC) spectra (Fig. 5) deduced the planar structure of 5, which is the same as the reported 3-acetyl-2-methyl-1,4,5-trihydroxy-2,3-epoxynaphthoquinol without determination of the absolute configuration [15]. In the present study, fine crystal from acetone was obtained and the absolute configuration of 5 was determined by single crystal X-ray diffraction (CDCC Number: 2158643) (Fig. 5). The result confirmed the planar structure of 5, and revealed unambiguously the absolute configuration as 1R, 2R, 3S, 4S.

Fig. 5
figure 5

1H–1H COSY, HMBC spectra and X-ray crystallographic structure of 5

Anti-fungal and anti-inflammatory inhibitory activity

Compounds 1 and 614 were evaluated for their inhibitory effects against three skin fungi (Epidermophyton floccosum, Trichophyton rubrum, Microsporum gypseum) at a concentration of 100 μM (Table S1), as previously described [28], with terbinafine as positive control. Most of them displayed only weak antifungal activity against the three skin fungi, while compound 9 showed obvious antifungal activities against E. floccosum and M. gypseum with MIC50 values of 2.467 ± 0.03 μM and 4.673 ± 0.077 μM, respectively. In case of the antifungal effects against E. floccosum and M. gypseum, simple emodin type anthraquinone 9 showed the strongest inhibition, followed by oxyglucoside anthraquinone (12) or C-glucoside oxanthrones (13, 14), and finally glycosylated seco analogues (1, 68). These compounds (1, 614) were also evaluated for their anti-inflammatory activity by using LPS to induce the production of iNOS from mouse monocyte macrophage RAW264.7, with L-NMMA as a positive control. The inhibition rate was shown in Table S2, all 10 anthraquinones showed NO inhibitory activity at a concentration of 50 μM, and the order of their inhibition rates is as follows: 9 > 11 > 12 > 13 > 14 > 8 > 10 > 1 > 6 > 7. Compound 9 had the strongest anti-inflammatory effect, followed by oxyglucoside anthraquinone, C-glucoside oxanthrones and finally seco-anthraquinone glucosides. It is noted that the anti-inflammatory and anti-fungal potential of emodin (9) decreased when it become glycosides or seco-anthraquinone cleavaged between C-10 and C-4a.

Experimental

General experimental procedures

UV spectra were given on a UV-2410PC Shimadzu spectrometer. One and two-dimensional NMR spectra were determined on acetone-d6 and methanol-d4 with Bruker Ascend-600 and AV-800 spectrometers. Chemical shifts (δ) were recorded in (parts per million, ppm) scale with TMS (Bruker, Zurich, Switzerland) as an internal standard. Coupling constants were expressed in hertz (Hz). ESI mass spectra were measured on a VG Auto Spec300 spectrometer. High-resolution electro-spray ionization mass (HRESIMS) spectra were performed on an API QSTAR Pular-1 spectrometer. Semi-preparative HPLC was performed on a Hanbon Sci & Tech with Capcell Pak Phenyl (250 mm × 10 mm, 5 μm) and Thermo Hypeersil GOLD aQ (250 mm × 9.4 mm × 5 μm) columns. Analytical HPLC was performed on a Waters 2695 Series HPLC system equipped with a reverse-phase ZORBAX SB-C-18 column (4.6 mm × 150 mm, 5 μm, Agilent Corporation, USA). Column chromatography (CC) was carried out using Sephadex LH-20 (25–100 μm, Pharmacia Fine Chemical Co., Ltd., Uppsala, Sweden), 75–100 μm MCI-gel CHP20P (Mitsubishi Chemical Co. Ltd., Tokyo, Japan), silica gel (100–200 mesh, Qingdao Marine Chemical, Inc., Qingdao, China) and macro-porous absorption resin (D101, Donghong Chemical Co., Ltd., People’s Republic of China). Acetonitrile (chromatographic grade) were purchased from XinLanJing (Pennsylvania, USA). Mouse mononuclear macrophage RAW264.7 was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, DMEM medium and fetal bovine serum were purchased from BI Company. Griess Reagent, LPS, Terbinafine hydrochloride, DMSO and control drug L-NMMA were purchased from Sigma. Epidermophyton floccosum, Trichophyton rubrum and Microsporum gypseum were purchased from the Medical Fungal Conservation Centre, Chinese Academy of Medical Sciences.

Plant materials

The roots of R. crispus were collected from Yimen town, Xianyang City, Shaanxi Province, in August 2020, and identified by Dr. En-De Liu from Kunming Institute of Botany (KIB), Chinese Academy of Sciences (CAS). A voucher specimen (KIBZL-20200803) is deposited at State Key Laboratory of Phytochemistry and Plant Resource in West China of KIB–CAS.

Extraction and isolation

The air-dried roots of R. crispus (10.0 kg) were crushed into small pieces and extracted with 90% aqueous MeOH at 60 °C (15 L × 4, each time 2 h). The organic solvent was removed under reduced pressure to yield a residue (1.6 kg), which was further extracted with ethyl acetate. After concentrated, the aqueous layer (480 g) was applied to a Sephadex LH-20 column chromatography (CC), eluting with water–methanol (1:0–0:1) to give two fractions (I–II). Fr. I (70 g) was subjected to CC over macroporous resin D101, eluting with H2O firstly to remove the sugars, and then with 100% MeOH. The yielded MeOH fraction (50.5 g) was subjected to CC over silica gel, eluting with a CHCl3/MeOH gradient system (1:0, 9:1, 8:2, 7:3, 6:4, 1:1, 0:1) to yield 6 fractions, A-F. Fr. A (10 g) was chromatographed on silica gel column with a petroleum ether/ethyl acetate gradient system (1:0, 9:1, 8:2, 7:3, 6:4, 1:1, 0:1) to yield 11 (4.8 mg), 10 (5.0 mg), 9 (100.0 mg). Fr. B (19.5 g) was appplied to RP-18 CC with a MeOH/H2O gradient system (from 0:1 to 1:0) to afford fractions B1-B4. Fr. B2 (500 mg) was purified by semipreparative HPLC (3 mL/min) with 7% MeCN/H2O (7:93) containing 0.1% trifluoroacetate to yield 1 (7.0 mg, retention time = 9 min), 6 (12.5 mg, retention time = 7 min), 7 (37.0 mg, retention time = 8 min), 8 (14.0 mg, retention time = 12 min), 12 (1.50 mg, retention time = 14 min), 13 (3.70 mg), 14 (17.0 mg). Fr. C (2.5 g) was separated by chromatography on a Chromatorex ODS column (2.5 cm i.d. × 35 cm) with 10 − 60% MeOH (5% stepwise, each 500 mL) to give Fr. C1 (50 mg) and Fr. C2 (1.0 g). Further, Fr. C1 was separated by semipreparative HPLC (Capcell Pak Phenyl, 250 mm × 10 mm × 5 μm, CH3CN/H2O 22:78) to obtain 2 (5.0 mg, retention time = 13.5 min) and 3 (12.0 mg, retention time = 17.3 min), and Fr. C2 was separated by semipreparative HPLC (Thermo Hypeersil GOLD aQ, 250 mm × 9.4 mm × 5 μm, CH3CN/H2O 28:72) to obtain 4 (50.0 mg, retention time = 8.0 min), 5 (305.0 mg, retention time = 10.0 min), respectively.

Crispuside A (1)

Yellowish amorphous powder; UV (MeOH) λmax (log ε) 206 (4.4), 274 (3.7) nm. 1H (600 MHz) and 13C (150 MHz) NMR (in methanol-d4) data, see Table 1. ESIMS m/z 449 [M−H]; HRESIMS m/z 449.1094 [M−H], (calcd C21H21O11: 449.1089).

Naphthalenone A (2)

White amorphous powder, \({\left[\alpha \right]}_{\mathrm{D}}^{19.5}\) + 15.03 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 222 (3.1) 270 (2.8), 330 (2.3) nm. 1H (600 MHz) and 13C (150 MHz) NMR (in methanol-d4) data, see Table 2. ESIMS m/z 235 [M−H]; HRESIMS m/z 235.0979 [M−H], (calcd C13H15O4, 235.0976).

Naphthalenone B (3)

White amorphous powder; \({\left[\alpha \right]}_{\mathrm{D}}^{19.5}\) + 1.58 (c 0.16, MeOH); UV (MeOH) λmax (log ε) 222 (3.1), 270 (2.8), 330 (2.3) nm. 1H (600 MHz) and 13C (150 MHz) NMR (in methanol-d4) data, see Table 2. ESIMS m/z 235 [M−H]; HRESIMS m/z 235.0979 [M−H], (calcd C13H15O4, 235.0976).

Naphthalenone C (4)

Yellow amorphous powder; \({\left[\alpha \right]}_{\mathrm{D}}^{25.9}\) + 13.43 (c 0.76, MeOH); UV (MeOH) λmax (log ε) 240 (3.1), 330 (2.6) nm. 1H (600 MHz) and 13C (150 MHz) NMR (in methanol-d4) data, see Table 2. ESIMS m/z 233 [M−H]; HRESIMS m/z 233.0814 [M−H], (calcd C13H13O4 233.0814).

(1R,2R,3S,4S)-3-Acetyl-2-methyl-1,4,5-trihydroxy-2,3-epoxynaphthoquinol (5)

White amorphous powder; 1H (500 MHz) and 13C (125 MHz) NMR (in methanol-d4) data, see Table 2. ESIMS m/z 249 [M−H].

Single-crystal X-ray diffraction data of 5

Colorless crystal of 5: C13H14O5, M = 250.24, a = 7.7634 (3) Å, b = 8.2971 (3) Å, c = 9.2218(3) Å, α = 90°, β = 107.7570 (10)°, γ = 90°, V = 565.71 (4) Å3, T = 100 (2) K, space group P1211, Z = 2, μ(Cu Kα) = 0.954 mm−1, 10,180 reflections measured, 2156 independent reflections (Rint = 0.0500). The final R1 values were 0.0319 [I > 2σ(I)]. The final wR(F2) values were 0.0821 [I > 2σ(I)]. The final R1 values were 0.0333 (all data). The final wR(F2) values were 0.0832 (all data). The goodness of fit on F2 was 1.064. Flack parameter = 0.17(10). Crystallographic data for the structure of 5 have been deposited in the Cambridge Crystallographic Data Centre (deposition number CCDC, 2,158,643). Copies of the data can be obtained free of charge from the CCDC via www.ccdc.cam.ac.uk.

The biological assay

The isolates 1 and 614 were evaluated for their anti-fungal against three skin fungi (Epidermophyton floccosum, Trichophyton rubrum, Microsporum gypseum) and anti-inflammatory activity. For anti-fungal activity, Terbinafine hydrochloride was used as positive control. Fungal broth (5 × 105 CFU mL−1) and test samples (100 μM) were incubated in 96-well plates at 25 °C for 5 days, A microplate reader was recorded by the absorbance at 625 nm. The experiment also set up the culture medium blank control, fungi control and terbinafine hydrochloride positive drug control.

The anti-inflammatory activity of 1 and 614 was screened as previously reported method [29]. The mouse mononuclear macrophage RAW264.7 was inoculated to 96 orifice plates and induced by 1.0 μg/mL LPS. At the same time, compounds 1 and 614 (final concentration 50 μM) was added, and no drug group and L-NMMA positive drug group were taken as controls. After the cells were cultured overnight, the medium was used to detect NO production and the absorbance was measured at 570 nm. MTS was added to the remaining medium to detect the cell survival rate and exclude the toxic effects of the compounds. The formula to calculate the inhibition rate is as follows: NO production inhibition rate (%) = (non-drug treatment group OD570 nm − sample group OD570 nm)/non-drug treatment group OD570 nm × 100%.

Conclusion

Four new (14) and ten known (514) quinone derivatives were isolated and identified from the roots of Rumex crispus L. Compound 1 is a seco-anthraquinone glucoside, while 24 belong to naphthalenones containing 3,4-dihydronaphthalen-1(2H)-one moiety. The absolute configuration of 5 was determined for the first time by X-ray single crystal diffraction. The anti-fungal and anti-inflammatory activity of anthraquinones (1, 614) was tested, of which compound 9 showed obvious anti-fungal activity. The results indicated that simple emodin type anthraquinone is more potential against skin fungi than its oxyglucosyl, C-glucosyl and glycosylated seco analogues.