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Abstract

Phytochemical investigation of the 70% ethanolic extract of the roots of Rubia oncotricha Hand.-Mazz. led to the isolation of 2 new anthraquinone glucosides named 1-hydroxy-2-hydroxymethyl-9,10-anthraquinone-11-O-β-D-glucopyranoside (1) and 1,3,6–trihydroxy-2-hydroxymethyl-9,10-anthraquinone-3-O-β-D-glucopyranoside (2), together with 5 known compounds (3-7). Their structures were elucidated by extensive spectroscopic data analysis (1-dimensional, 2-dimensional-nuclear magnetic resonance, and high resolution-electrospray ionization-mass spectrometry).

Keywords

folk herbs quinones structure elucidation

Introduction

Rubia oncotricha Hand.-Mazz., belonging to the plant family of Rubiaceae, is widely distributed in China as an endemic species. The dry roots and rhizomes of the plant have been used as Chinese folk herb for the treatment of cough, expectorant, jaundice, bronchitis, injuries, and so on by the local minorities in Guizhou province.^{1, 2} However, there have been few reports on the chemical constituents of this plant.

Previous phytochemical investigations of R oncotricha showed the isolation of anthraquinones, naphthoquinones, naphthohydroquinones, and triterpenoids,^3–5 some of which exhibited biological activities, such as antitumor and antinematodal activities.^{6, 7} During the course of searching for bioactive compounds, we performed a phytochemical study on the root and rhizome of R oncotricha. Previous studies mainly focused on the isolation and identification of low polar compounds, and there have been few reports on the highly polar chemical constituents of R oncotricha. Therefore, an n-butanol fraction of 70% EtOH extract of this species was investigated, which led to the isolation of 2 new anthraquinone glucosides (1-2) together with 5 known compounds (3-7) (Figure 1). Compounds (3-7) were isolated from genus Rubia for the first time. Herein, we described the structure elucidation of these new compounds.

Figure 1.

Chemical structures of compounds 1 to 7 isolated from Rubia oncotricha.

Results and Discussion

Compound 1 was obtained as a yellow amorphous powder. The molecular formula of 1 was established as C₂₁H₂₀O₉ by its high-resolution-electrospray ionization-mass spectrometry (HR-ESI-MS) at m/z 415.1025 [M-H]⁻ (calcd for C₂₁H₁₉O₉, 415.1023). The infrared (IR) spectrum showed absorption bands of hydroxyl (3293 cm⁻¹), phenyl (2870 and 1591 cm⁻¹), and carbonyl (1669 and 1633 cm⁻¹) groups. The ¹H nuclear magnetic resonance (NMR) spectrum (Table 1) showed signals for 1 unsubstituted anthraquinone aromatic ring [δ_H 8.24 (1H, m, H-8), 8.19 (1H, m, H-5), and 7.95 (2H, overlap, H-6, 7)], which is a 1,2-disubstituted benzene. Signals at δ_H 8.04 (1H, d, J = 7.6 Hz, H-3) and 7.74 (1H, d, J = 7.6 Hz, H-4) for 2 ortho-coupled protons suggested a disubstituted anthraquinone aromatic ring. One oxygen-bearing methylene proton signals at δ_H 4.91 (1H, d, J = 15.2 Hz, H-11a) and 4.76 (1H, d, J = 15.2 Hz, H-11b) and 1 anomeric proton signal of glucose at δ_H 4.34 (1H, d, J = 7.6 Hz, H-1′). The ¹³C NMR spectrum (Table 1) showed 21 carbon signals, including 2 carbonyl carbons [δ_C 188.6 (C-9) and 181.8 (C-10)], 12 aromatic carbons [δ_C 158.5 (C-1), 135.3 (C-3), 134.7 (C-7), 134.6 (C-6), 134.1 (C-2), 133.2 (C-8a), 132.8 (C-10a), 131.8 (C-4a), 126.9 (C-5), 126.6 (C-8), 118.7 (C-4), 115.2 (C-9a)], 1 oxygen-bearing methylene carbon [δ_C 64.0 (C-11)], and 6 carbons from glucopyranosyl moiety [δ_C 102.7 (C-1′), 77.1 (C-3′), 76.7 (C-5′), 73.6 (C-2′), 70.1 (C-4′), 61.1 (C-6′)]. Together, these data suggested that compound 1 revealed a structure of anthraquinone glucoside. The β-configuration of the sugar unit was assigned by high coupling constants (J = 7.6 Hz) of the anomeric proton. The absolute configuration of glucose was determined as D by gas chromatography (GC) analysis of its derivative of hydrolysate. In the ¹H detected heteronuclear multiple bond correlation (HMBC) spectrum, the correlations from H-11 to C-1, C-2, and C-1′ and from H-3 to C-1 and C-11 verified that the glucopyranosyl moiety was connected to C-11 (Figure 2). Therefore, the structure of 1 was assigned as 1-hydroxy-2-hydroxymethyl-9,10-anthraquinone-11-O-β-D-glucopyranoside.

Figure 2.

Key HMBC correlations of 1 and 2.

Table 1.

¹H and ¹³C NMR Spectroscopic Data of Compounds 1 and 2 in DMSO-d₆ (δ in ppm, J in Hz).

No.	1^a		2^a
No.	δ _H	δ _C	δ _H	δ _C
1		158.5		161.6
2		134.1		123.8
3	8.04 (d, 7.6)	134.6		161.8
4	7.74 (d, 7.6)	118.6	7.42 (s)	106.1
4a		131.8		133.8
5	8.19 (m)	126.9	7.48 (d, 2.4)	112.9
6	7.95 (overlap)	135.2		164.3
7	7.95 (overlap)	134.7	7.24 (dd, 2.4, 8.8)	121.7
8	8.24 (m)	126.6	8.09 (d, 8.8)	129.7
8a		133.2		124.1
9		188.6		186.3
9a		115.2		111.1
10		181.8		181.7
10a		132.8		135.3
11	4.91 (d, 15.2)	64.0	4.62 (d, 11.2)	50.9
	4.76 (d, 15.2)		4.54 (d, 11.2)
1′	4.34 (d, 7.6)	102.7	5.06 (d, 7.6)	100.9
2′	3.10 (overlap)	73.6	3.29 (overlap)	73.4
3′	3.14 (overlap)	77.1	3.41 (overlap)	77.4
4′	3.08 (overlap)	70.0	3.19 (overlap)	69.4
5′	3.18 (overlap)	76.7	3.32 (overlap)	76.0
6′	3.46 (dd, 6.0, 11.6)	61.0	3.55 (dd, 5.2, 11.8)	60.4
	3.70 (dd, 5.6, 11.6)		3.71 (dd, 5.0, 11.8)

Abbreviations: NMR, nuclear magnetic resonance; DMSO-d₆, dimethylsulfoxide-d₆

¹H at 400 MHz and ¹³C at 100 MHz in DMSO-d₆.

Compound 2 was isolated as a yellow amorphous powder. The molecular formula of 2 was established as C₂₁H₂₀O₁₁ by its HR-ESI-MS at m/z 447.0920 [M-H]⁻ (calcd for C₂₁H₁₉O₁₁, 447.0921). The IR spectrum showed absorption bands of hydroxyl (3455 cm⁻¹), phenyl (3196 and 1598 cm⁻¹), and carbonyl (1662 cm⁻¹) groups. The ¹H NMR spectrum (Table 1) showed 1 ABX coupling system anthraquinone aromatic benzene [δ_H 8.09 (1H, d, J = 8.8 Hz, H-8), 7.48 (1H, d, J = 2.4 Hz, H-5), and 7.24 (1H, dd, J = 2.8, 8.8 Hz, H-7)], 1 isolated aromatic proton [δ_H 7.42 (1H, s, H-4)]. One hydroxymethyl proton signal [δ_H 4.62 (1H, d, J = 11.2 Hz, H-11a), 4.54 (1H, d, J = 11.2 Hz, H-11b)] and an anomeric proton signal of sugar unit [δ_H 5.06 (1H, d, J = 7.6 Hz, H-1′)]. The ¹³C NMR spectrum (Table 1) showed 21 carbon signals, including 2 carbonyl carbons [δ_C 186.3 (C-9) and 181.7 (C-10)], 12 aromatic carbons [δ_C 164.3 (C-6), 161.8 (C-3), 161.6 (C-1), 135.3 (C-10a), 133.8 (C-4a), 129.7 (C-8), 124.1 (C-8a), 123.8 (C-2), 121.7 (C-7), 112.9 (C-5), 111.1 (C-9a), 106.1 (C-4)], 1 hydroxymethyl carbon [δ_C 50.9 (C-11)], and 6 carbons from glucopyranosyl moiety [δ_C 100.9 (C-1′), 77.4 (C-3′), 76.0 (C-5′), 73.4 (C-2′), 69.4 (C-4′), 60.4 (C-6′)]. The above data obtained indicated that 2 was also an anthraquinone glucoside derivative and the 1-dimensional (1D)-NMR data of 2 was similar to 1,3,6-trihydroxy-2-hydroxymethyl-9,10-anthraquinone-3-O-(6′-O-acetyl)-β-D-glucopyranoside by comparison of their NMR data.⁸ The C-6′ signal of 2 (at δ_C 60.4) was shifted downfield to δ_C 63.4 in 1,3,6-trihydroxy-2-hydroxymethyl-9,10-anthraquinone-3-O-(6′-O-acetyl)-β-D-glucopyranoside due to the C-6′ acetyl group. The configuration of the anomeric proton of glucose was proposed as β on the basis of its coupling constant (J = 7.6 Hz). The absolute configuration of glucose was determined as D by GC analysis of its derivative of hydrolysate. In the HMBC spectrum, the glucose moiety was determined to be bound to C-3 of the anthraquinone based on the correlations between H-1′/C-3 and H-4/ C-2, C-3 (Figure 2). Additional HMBC correlation peaks were also observed between H-11/C-1, H-11/C-2, H-5/C-6, H-5/C-7, H-7/C-5, H-8/C-6, and H-8/C-9. These results confirmed that 2 hydroxyl groups were present at C-1 and C-6, and 1 hydroxymethyl was present at C-2. Thus, compound 2 was elucidated as 1,3,6-trihydroxy-2-hydroxymethyl-9,10-anthraquinone-3-O-β-D-glucopyranoside.

In addition, 5 known compounds (3-7) were identified by comparing the measured spectroscopic data with the reported in the literature, including 3 lignins, threo-dihydroxydehy-drodiconiferyl alcohol (3),⁹ (7R,8R)-7,8-dihydro-9′-hydroxyl-3′-methoxyl-8-hydroxymethyl-7-(4-hydroxy-3-methoxyphenyl)-1′-benzofuranpropanol 9′-O-β-D-glucopyranoside (6)¹⁰ and (7S,8R)-dihydrodehydrodiconiferyl alcohol-9-O-β-D-glucopyranoside (7),¹¹ 1 phenolic glycoside, 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (4),¹² and 1 monoterpene glycoside, (–)-angelicoidenol-2-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (5).^{13, 14} These compounds (3-7) were isolated from genus Rubia for the first time.

Conclusions

In the present work, 2 new anthraquinone glucosides (1-2), along with 5 known compounds (3-7) were isolated and identified from the roots of R oncotricha and characterized using 1D and two-dimensional (2D)-NMR and HR-ESI-MS spectroscopic analysis. Compounds 3-7 were isolated from the plants of genus Rubia for the first time. The current study provided more information on the chemical composition of R oncotricha, which lays a foundation for the development of this plant used as a Chinese folk herb.

Experimental

General

HR-ESI-MS was measured on a Bruker Daltonics micro TOF-Q II mass spectrometer (Bruker Daltoniks GmbH). 1D and 2D NMR were recorded on a JEOL ECS 400 NMR spectrometer (Jeol). Ultraviolet (UV) spectra were acquired on a UV-2700 spectrometer (Shimadzu Corporation). Optical rotation values were measured on an AUTOPOLⅥ spectrometer (Rudolph Co., Ltd). IR spectra were recorded on an IR Tracer-100 spectrometer (Shimadzu Corporation). Column chromatography was performed with macroporous resin (D101, Tianjin Haiguang Chemical Co., Ltd), silica gel (200-300 mesh and 300-400 mesh, Qingdao Haiyang Chemical Co., Ltd), Toyopearl HW-40C (Tocho Corporation), Toyopearl HW-40F (Tocho Corporation) and Sephadex LH-20 (Pharmacia Biotech). Semipreparative high-performance liquid chromatography (HPLC) was performed on Shimadzu LC-20AP with ACE C18-PFP columns (10 × 250 mm, 5 μm) (Phenomenex). Thin layer chromatography was performed on silica gel GF 254 plates (Qingdao Haiyang Chemical Co. Ltd). GC analysis was carried out on a Shimadzu-2010 plus gas chromatograph (Shimadzu Corporation) using a ZB-5 capillary column (30 m × 0.25 mm i.d. × 0.25 μm).

Plant Material

The roots and rhizomes of R oncotricha were purchased in September 2018 in Guiyang, Guizhou Province, China, and were identified by Associate Professor Qingde Long from the Department of Medicinal Botany and Pharmacognosy (School of Pharmacy, Guizhou Medical University). The specimen (No. 20180227) was stored at the Guizhou Provincial Key Laboratory of Pharmaceutical Preparations at Guizhou Medical University.

Extraction and Isolation

The dried root and rhizome of R oncotricha (5.0 kg) were extracted with 70% (v/v) EtOH 3 times. After evaporation of the solvent under reduced pressure, the crude extract (592.0 g) was dissolved in water and then partitioned in turn with petroleum ether, ethyl acetate, and n-butanol. The n-butanol was evaporated under reduced pressure to yield 215.0 g. The n-butanol extract (215.0 g) was chromatographed on silica gel column chromatography eluted gradient with a gradient of CHCl₂-MeOH (30:1, 20:1, 15:1, 10:1, 7:1, 4:1, 1:1, v/v) to give 11 fractions (Fr.1-11).

Fraction Fr.2 (44.1 g) was subjected to silica gel column chromatography eluting with EtOAc-MeOH (15:1 to 1:1, v/v) afforded 9 subfractions (Fr.2.1-2.9), Fr.2.2 (2.7 g) was fractionated by silica gel column chromatography eluting with EtOAc and Toyopearl HW-40F column chromatography (MeOH) to yield compound 4 (50.0 mg). Fr.2.5 (18.4 g) was subjected to silica gel column chromatography eluting with EtOAc-MeOH (5:1, v/v) afforded 5 subfractions (Fr.2.5.1-2.5.5), Fr.2.5.2 (1.5 g) was further subjected by repeated Sephadex LH-20 column chromatography (MeOH), Toyopearl HW-40F column chromatography (MeOH), and silica gel column chromatography (CHCl₃-MeOH solvent system from 6:1 to 4:1, v/v) to yield compound 5 (12.4 mg).

Fr.3 (2.5 g) was subjected to silica gel column chromatography eluted with EtOAc-MeOH (8:1-1:1, v/v) to obtain 7 subfractions (Fr.3.1-3.7). Fr.3.5 (334.8 mg) was further subjected to repeated Sephadex LH-20 column chromatography (MeOH), and Toyopearl HW-40F column chromatography (MeOH) to obtain Fr.3.5.1(18.0 mg). Further purification of Fr.3.5.1 with preparative HPLC (1.5 mL/min, MeOH/H₂O 56:44, v/v) yielded compounds 6 (3.2 mg) and 7 (4.1 mg).

Fr.4 (10.72 g) purified by Sephadex LH-20 column chromatography (MeOH) afforded 6 subfractions (Fr.4.1-4.6). Fr.4.3 (2.4 g) was applied to MCI column chromatography (50%-100% MeOH/H₂O, v/v) and Toyopearl HW-40F column chromatography (MeOH) to get compound 3 (4.0 mg). Fr.4.4 (1.8 g) was further subjected to repeated Toyopearl HW-40F column chromatography (MeOH) and MCI column chromatography (50%-100% MeOH/H₂O, v/v) to obtain compound 2 (9.0 mg).

Fr.6 (14.0 g) was subjected to silica gel column chromatography eluted with EtOAc-MeOH (15:1-1:1, v/v) to obtain 5 subfractions (Fr.6.1-6.5). Fr.6.3 was separated by repeated Sephadex LH-20 column chromatography (CHCl₃/MeOH 1:1) and Sephadex LH-20 column chromatography (MeOH) to Fr.6.3.1. Fr.6.3.1 (95.0 mg) was subjected to silica gel column chromatography eluted with EtOAc-MeOH (15:1-1:1, v/v) and Toyopearl HW-40F column chromatography (MeOH) to obtain compound 1 (10.0 mg).

1-Hydroxy-2-hydroxymethyl-9,10-anthraquinone-11-O-β-D-glucopyranoside (1) yellow amorphous solid; $[α]_{D}^{20}$ −37.2 (c 0.19, MeOH); UV (MeOH) λ_max (log ε): 199.5 (4.41), 223.5 (4.25), 253.5 (4.43) nm; HR-ESI-MS m/z: 415.1025 [M-H]⁻ (calcd for C₂₁H₁₉O₉: 415.1023);¹H NMR (dimethylsulfoxide-d₆ [DMSO-d₆], 400 MHz) and ¹³C NMR (DMSO-d₆, 100 MHz) spectral data: see Table 1.

1,3,6-Trihydroxy-2-hydroxymethyl-9,10-anthraquinone-3-O-β-D-glucopyranoside (2) yellow amorphous solid; $[α]_{D}^{20}$ −14.1 (c 0.13, MeOH); UV (MeOH) λ_max (log ε): 204.0 (4.31), 218.5 (4.40), 270.0 (4.18) nm; HR-ESI-MS m/z: 447.0920 [M-H]⁻ (calcd for C₂₁H₁₉O₁₁: 447.0921); ¹H NMR (DMSO-d₆, 400 MHz) and ¹³C NMR (DMSO-d₆, 100 MHz) spectral data: see Table 1.

Compound 3 $[α]_{D}^{20}$ +15.3 (c 0.48, MeOH), compound 4 $[α]_{D}^{20}$ +2.1 (c 3.90, MeOH), compound 5 $[α]_{D}^{20}$ −15.4 (c 1.30, MeOH), compound 6 $[α]_{D}^{20}$ −20.4 (c 0.39, MeOH), compound 7 $[α]_{D}^{20}$ −35.8 (c 0.67, MeOH), the ¹H NMR, ¹³C NMR, UV and IR spectral data of compounds 3-7 are presented in Supplemental material.

Acid Hydrolysis and Determination of Absolute Sugar Configuration

Each compound (0.5 mg) was hydrolyzed with 2 M HCl (2.0 ml) at 95 °C for 3 h. After cooling, the reaction mixture was partitioned between water and ethyl acetate 3 times. The aqueous layer was repeatedly evaporated to dryness with methanol, until neutral. The residue was dissolved in pyridine (0.4 mL), then L-cysteine methyl ester hydrochloride (1.0 mg) was added. The mixture was reacted at 60 °C for 1 h, and then trimethylsilyl imidazole (0.15 mL) was added to the reaction mixture at 60 °C for another 1 h, after the reaction was completed, the reaction solution was blown dry with nitrogen. The reaction residue was dissolved in water (1.0 mL), and then n-hexane (0.5 mL) was added for extraction 3 times, and the n-hexane layer was analyzed by GC. The absolute configurations of the monosaccharides were confirmed to be D-glucose by comparison of the retention times of the authentic sample [t_R (D-glucose) 29.196 min, t_R(L-glucose) 29.558 min)] (Supplemental material).

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221117311 - Supplemental material for Chemical Constituents From the Roots of Rubia oncotricha

Supplemental material, sj-docx-1-npx-10.1177_1934578X221117311 for Chemical Constituents From the Roots of Rubia oncotricha by Li Jiang, Zhilong He, Yushan Nie, Yang Wang, Xue Ma, Ting Liu, Yuan Lu, Yonglin Wang and Yongjun Li in Natural Product Communications

Footnotes

Acknowledgements

We are grateful to Associate Professor Qingde Long from the Department of Medicinal Botany and Pharmacognosy (School of Pharmacy, Guizhou Medical University) for identifying plant species.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Statement of Human and Animal Rights

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

Statement of Informed Consent

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

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China (U1812403), Central Government Guides Local Science and Technology Special Projects (20184006), and Innovation Talent Team Project of Guizhou Province ( 20165677).

Ethical Approval

Ethical Approval is not applicable for this article.

ORCID iDs

Li Jiang

Zhilong He

Supplemental Material

Supplemental material for this article is available online.

References

Editorial Committee of Chinese Flora of Chinese Academy of Sciences. Flora of China. Vol. 71. China Science Publishing ＆ Media Ltd; 1999.

Guizhou Medical Products Administration. Quality standards of traditional Chinese medicine and ethnic medicine in Guizhou Province. 2003. Guizhou Science and Technology Publishing House Co., Ltd; 2003.

Qiao

Takeya

Itokawa

Iitaka

. Three novel naphthohydroquinone dimers from Rubia oncotricha. Chem Pharm Bull. 1990;38(10):2896‐2898. doi:10.1248/cpb.38.2896

Itokawa

Qiao

Takeya

. Anthraquinones, naphthquinones and naphthohydroquinones from Rubia oncotricha. Phytochemistry. 1991;30(2):637‐640. doi:10.1016/0031-9422(91)83742-4

Itokawa

Qiao

Takeya

. New arborane type triterpenoids from Rubia cordifolia var. Pratensis and R. oncotricha. Chem Pharm Bull. 1990;38(5):1435‐1437. doi:10.1248/cpb.38.1435

Zhao

Wang

Chen

Huang

Tan

. (±)-Rubioncolin D, a pair of enantiomeric naphthohydroquinone dimers from Rubia oncotricha. Tetrahedron Lett. 2017;58(31):3041‐3043. doi: 10.1016/j.tetlet.2017.06.063

Wang

Zhao

Zeng

Tan

. Chemical constituents from roots and rhizomes of Rubia oncotricha and their cytotoxic activities. Chin J Chin Mater Med. 2018;43(22):4462‐4468. doi: 10.19540/j.cnki.cjcmm.2018.0119

Fan

Kuang

Zeng

, et al. Biologically active arborinane-type triterpenoids and anthraquinones from Rubia yunnanensis. J Nat Prod. 2011;74(10):2069‐2080. doi: 10.1021/np2002918.

Wang

Yang

Khan

Liu

Wang

. Neolignans, lignans and glycoside from the fruits of Melia toosendan. Fitoterapia. 2014;99:92‐98. doi: 10.1016/j.fitote.2014.09.008

10.

Dong

Chen

Liu

. A new neolignan glycoside from the leaves of Acer truncatum. Molecules. 2006;11(12):1009‐1014. doi:10.3390/11121009

11.

Tang

, et al. Lignan glycosides from Neoalsomitra integrifoliola. J Nat Prod. 2008;71(5):784‐788. doi:10.1021/np070565+

12.

Shao

Wang

Huang

Sang

. Stilbene glucoside from Polygonum multiflorum Thunb.: a novel natural inhibitor of advanced glycation end product formation by trapping of methylglyoxal. J Agric Food Chem. 2010;58(4):2239‐2245. doi: 10.1021/jf904122q

13.

Yan

Dong

Liu

Chao

Hao

. Study on chemical constituents of Asterageratoides var. polosu. J Xinxiang Med Coll. 2007;24(6):548‐550. doi:10.3969/j.issn.1004-7239.2007.06.003

14.

Inoshiri

Saiki

Kohda

Otsuka

Yamasaki

. Monoterpene glucosides from Berchemza racemosa. Phytochemistry. 1988;27(9):2869‐2871. doi: 10.1016/0031-9422(88)80678-2

Supplementary Material

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