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Abstract

We describe here the isolation of 1 previously unreported neolignan (7S,8R)-2′,3-dimethoxy-4′,7-epoxy-8,5′-neolignan-4,9,9′-triol-9′-O-β-d-glucopyranoside (1) and 4 flavonoid glycosides, quercetin 7-O-glucoside (2), Isoquercitrin (3), quercetin 3-O-rutinoside (4), narcissin (5), as well as 2 tannins, isomallotusinin (6), isocorilagin (7), from Erodium oxyrrhynchum M. Bieb. Structures were determined using 1-dimensional and 2-dimensional-nuclear magnetic resonance, liquid chromatography-mass spectrometry, infrared and ultraviolet spectroscopy, while stereochemistry was confirmed by circular dichroism and optical rotation.

Keywords

neolignan tannin flavonoid

Introduction

Erodium oxyrrhynchum M. Bieb. is an annual herbaceous plant belonging to the genus Erodium L’Herit (Geraniaceae). The genus Erodium comprises about 60 species and is mainly distributed in Mediterranean and Western Asian regions.¹ While the chemistry of several Erodium species has been reported previously, this is the first investigation of E. oxyrrhynchum. Various parts of Erodium species have been used medicinally as an astringent, hemostatic, diaphoretic, diuretic, anti-inflammatory, topical disinfectant, and to increase lactation.² The range of ethnomedical uses of this genus and the limited knowledge of phytochemical constituents of this species make it an attractive target for further investigation.

Results and Discussion

Compound 1 was obtained as a yellowish amorphous powder; [ɑ ]¹⁸ _D −43.06 (c 0.2, methanol [MeOH]); UV λ _max (MeOH) nm (logε) 211 (4.31), 231 (3.96), and 282 (3.59). The molecular formula C₂₆H₃₄O₁₁ with 10 degrees of unsaturation was determined based on a high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) pseudomolecular ion at m/z 545.1989 ([M + Na] ⁺, calcd 545.1999). In the infrared spectrum, absorption bands for hydroxy (3358 cm⁻¹) and aromatic ring (1602 cm⁻¹) groups were observed. The ¹H-nuclear magnetic resonance (NMR) spectrum revealed the presence of a 1,3,4-trisubstituted aromatic ring (δ_H 6.75 [1H, d, J = 8.0 Hz, H-5], 6.81 [1H, dd, J = 8.0, 1.9 Hz, H-6], 6.94 [1H, d, J = 1.9 Hz, H-2]), a 1,2,4,5-tetrasubstituted aromatic ring (6.74 [2H, s, H-3′, H-6′])], hydroxypropyl protons at δ _H 1.89 (2H, m, H₂-8′), 2.65 (2H, t, J = 7.5 Hz, H₂-7′), 3.52 (1H, dt, J = 9.6, 6.3 Hz, H-9′b), and 3.92 (1H, dt, J = 9.6, 6.3 Hz, H-9′a). Significant correlations between H-8′/H-7′ and H-8′/H-9′ can be seen in the ¹H–¹H-correlation spectroscopy (COSY) spectrum. Also observed in the ¹H-NMR spectrum are hydroxymethyl protons δ _H 3.75 (1H, dd, J = 11.2, 7.2 Hz, H-9b), 3.82 (1H, dd, J = 11.2, 6.2 Hz, H-9a), an oxygenated methine proton at δ _H 5.48 (1H, d, J = 6.3 Hz, H-7), a methine proton at δ _H 3.46 (1H, m, H-8), 2 methoxyl resonances at δ _H 3.80 (3H, s, OCH₃), 3.84 (3H, s, OCH₃), in addition to a glucosyl anomeric proton at δ _H 4.24 (1H, d, J = 7.8 Hz, H-1″). The conﬁguration of the glucosidic linkage was determined to be β based on the coupling constant (J = 7.8 Hz). Signiﬁcant heteronuclear multiple bond correlation (HMBC) correlations (shown in Figure 1) were observed between H-7/C-5′ and H-8/C-4′. Therefore, it could be concluded that 2 phenylpropanoids formed a 7,8-dihydro-8-hydroxymethyl-7-phenyl-1′-benzofuranpropanol skeleton. The HMBC correlation between H-1″ and C-9′ indicated that the glucopyranosyl moiety was connected at C-9′. The relative conﬁguration of H-7 and H-8 was established as trans based on the chemical shift of C-8 (δ 55.4).³ The absolute configurations of dihydrobenzofuran neolignans are usually determined by the signs of the band ¹L_b (270, 300 nm) or ¹L_a (220, 240 nm) in the circular dichroism (CD) spectrum.⁴ The positive sign of the ¹L_b band predicts the absolute configuration of 7,8-trans-3-methoxydihydrobenzofuran neolignans to be (7S, 8R). The CD spectrum of 1 showed a positive Cotton effect at 289 nm (∆ℇ +0.43), indicating that the absolute configuration of 1 was the (7S, 8R)-configuration. Therefore, compound 1 was elucidated as (7S,8R)−2',3-dimethoxy-4′,7-epoxy-8,5′-neolignan-4,9,9′-triol-9′-O-β-d-glucopyranoside. The structure of compound 1 is shown in Figure 2.

Figure 1

Key heteronuclear multiple bond correlations of compound 1.

Figure 2

The structures of compounds 1-7.

The other isolated compounds were determined to be quercetin 7-O-glucoside (2),⁵ isoquercitrin (3),⁶ quercetin 3-O-rutinoside (4),⁷ narcissin (5),⁸ isomallotusinin (6),⁹ isocorilagin (7)⁹ by comparison of their NMR spectral data with those reported in the literature (copies of all spectroscopic data are included in Supplemental Material 1). The structures of all the isolated compounds are shown in Figure 2.

Compounds 1-7 were evaluated for cytotoxicity against human breast adenocarcinoma cancer cell line (MCF-7) using a standard 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyl tetrazolium bromide (MTT) assay. While compounds 1-5 were inactive in this assay, compound 6 showed weak cytotoxicity against MCF-7 cell line, with a half-maximal inhibitory concentration (IC₅₀) of 256.30 µM, and compound 7 showed moderate cytotoxicity with an IC₅₀ of 41.31 µM.

Experimental

General

Column chromatography was performed on Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) or Diaion HP-20 (Mitsubishi Chemical Corp., Tokyo, Japan). Flash chromatography was performed on an ISCO CombiFlash Rf+ instrument (Teledyne ISCO, Lincoln NE, USA) using prepacked columns of spherical C18 bonded silica 20–45 µm (SepaFlash, Santai Technologies, Changzhou, China). Analytical high performance liquid chromatography (HPLC) separations were performed on an Agilent 1260VL quad gradient system (G1311C pump, G1329B autosampler, G1316A thermostatted column compartment, and G1315D photodiode array detector, Agilent Technologies, Santa Clara CA, USA), using a Hypersil Gold C18 analytical column (3 µm, 2.1 × 150 mm, Thermo Scientific, Waltham MA, USA). Preparative HPLC separations were performed on an Agilent 1260VL quad gradient system (G1311C pump, and G1315D photodiode array detector) equipped with a Rheodyne 7725i manual injection valve (IDEX Health & Science, Middleboro MA, USA) and Shimadzu CTO-20 A column oven (Shimadzu Scientific Instruments, Kyoto, Japan). Preparative separations were conducted using a Hypersil Gold C18 column (5 µm, 21.2 × 250 mm). Mass spectra were determined on a Thermo Fisher Q Exactive Orbitrap LC-MS. NMR spectra were recorded on Bruker Avance III spectrometers (Bruker Biospin Corp., Billerica MA, USA) operating at 600 MHz (¹H) and 150 MHz (¹³C). Residual solvent resonances were used as an internal reference, and chemical shifts are reported in ppm (δ). Optical rotations were determined with an Autopol-II polarimeter (Rudolf Research Analytical, Hackettstown NJ, USA). Circular dichroism studies were performed using a Bio-Logic SAS MOS-500 instrument (Bio-Logic SAS, Claix, France).

Plant Material

Samples comprising the aerial portions of E. oxyrrhynchum M.Bieb. were collected near Nubarashen village in the Artashat district of Ararat Province, Armenia (lat. 40.10, long. 44.55, elev. 1190 m) in late May 2006. Specimens documenting the collection, Fayvush and Tamanyan 18–2006, have been deposited in the herbaria of the Armenian National Academy of Sciences (ERE) and the New York Botanical Garden (NY, barcode 3350475). The collected plant material was freed of soil and other extraneous material, air-dried in the shade, and then milled to a coarse powder.

Extraction and Isolation

The dried and powdered aerial parts of E. oxyrrhynchum (1 kg) were extracted with MeOH (3 × 6 L) at ambient temperature, and the combined MeOH extracts were concentrated in vacuo to yield a tarry residue (95.0 g). The resulting residue was suspended in 90% (aqueous) MeOH (250 mL) and partitioned with n-hexane (3 × 200 mL). The defatted MeOH fraction was freed of solvent in vacuo, dispersed in water (250 mL), and extracted successively with dichloromethane and n-BuOH (each 3 × 200 mL) to obtain a gross separation into hexane, dichloromethane, butanol, and water-soluble fractions.

The n-BuOH fraction (31.57 g) was chromatographed on the macroporous resin, Diaion HP20 (450 g), eluted with water, 20%, 40%, 60%, 80%, and 100% aqueous MeOH (1000 mL each). Six fractions (Frs. 1-6) were obtained after the evaporating solvent.

Fraction 3, eluted with 40% MeOH, was purified by preparative HPLC to give compound 7 (45.9 mg). On analytical HPLC, a retention time of 8.9 minutes was observed for compound 7 (Hypersil Gold C18, acetonitrile:water [ACN:H₂O, 11:89] containing 0.1% formic acid, 0.2 mL/min, 40°C).

Fraction 5, eluted with 60% MeOH, was analyzed by HPLC then fractionated by flash chromatography on a C18 column, eluting with ACN and H₂O (1:3) to give 4 subfractions (A-D).

Fraction A was purified by preparative HPLC to yield compound 4 (47.9 mg). On analytical HPLC, a retention time of 17.8 minutes was observed for compound 4 (Hypersil Gold C18, ACN:H₂O [12:88] containing 0.1% formic acid, 0.2 mL/min, 40°C).

Fraction C was purified by preparative HPLC to yield compound 2 (40.3 mg). On analytical HPLC, a retention time of 23.3 minutes was observed for compound 2 (Hypersil Gold C18, ACN:H₂O [14:86] containing 0.1% formic acid, 0.2 mL/min, 40°C).

Fraction B was subjected to chromatography on Sephadex LH-20, eluting with MeOH followed by MeOH–H₂O (1:1) to give 6 subfractions (B1-B6).

Fraction B1 was purified by preparative HPLC to give compound 1 (5.9 mg). On analytical HPLC, a retention time of 17.2 minutes was observed for compound 1 (Hypersil Gold C18, ACN:H₂O [15:85] containing 0.1% formic acid, 0.2 mL/min, 40°C).

Fraction B3 was purified by preparative HPLC to give compound 3 (15.7 mg). On analytical HPLC, a retention time of 15.6 minutes was observed for compound 3 (Hypersil Gold C18, ACN:H₂O [13:87] containing 0.1% formic acid, 0.2 mL/min, 40°C).

Fraction B2 was purified by preparative HPLC to give compound 5 (8.3 mg). On analytical HPLC, a retention time of 16.4 minutes was observed for compound 5 (Hypersil Gold C18, ACN:H₂O [15:85] containing 0.1% formic acid, 0.2 mL/min, 40°C).

Fraction B4 was purified by preparative HPLC to give compound 6 (5.9 mg). On analytical HPLC, a retention time of 23.4 minutes was observed for compound 6 (Hypersil Gold C18, ACN:H₂O [14:86] containing 0.1% formic acid, 0.2 mL/min, 40°C).

(7S, 8R)-2′,3-Dimethoxy-4′,7-Epoxy-8,5′-Neolignan-4,9,9′-triol-9′-O-β-d-Glucopyranoside (1)

Yellowish amorphous powder.

¹H-NMR and ¹³C-NMR (deuterated methanol): Table 1.

Table 1

¹H (500 MHz) and ¹³C (125 MHz) Nuclear Magnetic Resonance Data for Compound 1 in Deuterated Methanol.

No.	δ_C	δ_H (mult., J in Hz)	No.	δ_C	δ_H (mult., J in Hz)
1	134.8	-	5′	147.4	-
2	110.5	6.94 (1H, d, 1.9)	6′	114.2	6.74 (1H, s)
3	149.0	-	7′	32.8	2.65 (2H, t, 7.5)
4	147.5	-	8′	32.9	1.89 (2H, m)
5	116.1	6.75 (1H, d, 8.0)	9′	69.9	3.52 (1H, dd, 9.6, 6.3) 3.92 (1H, dd, 9.6, 6.3)
6	119.7	6.81 (1H, dd, 1.9, 8.0)	1″	104.5	4.24 (1H, d, 7.8)
7	88.9	5.48 (1H, d, 6.3)	2″	75.1	3.19 (1H, dd, 8.7, 7.8)
8	55.4	3.46 (1H, m)	3″	78.1	3.30 (1H, d, 8.7, 8.6)
9	64.9	3.75 (1H, dd, 11.2, 7.2) 3.82 (1H, dd, 11.2, 6.2)	4″	71.6	3.27 (1H, d, 8.6)
1’	136.8	-	5″	77.9	3.24 (1H, dd, 5.5, 2.2)
2’	145.1	-	6″	62.7	3.65 (1H, dd, 11.8, 5.5) 3.85 (1H, dd, 11.8, 2.2)
3’	118.0	6.74 (1H, s)	OCH₃	56.3	3.80 (3H,s)
4’	129.8	-		56.7	3.84 (3H,s)

HR-ESI-MS: m/z 545.1989 [M + Na⁺] (calcd for C₂₆H₃₄NaO₁₁, 545.1999).

Acid Hydrolysis of Compound 1

Compound 1 (2 mg) was dissolved in 1 M hydrochloric acid (dioxane:H₂O [1:1], 6 mL) and heated in a water bath for 2 hours.¹⁰ After the solvent was removed in vacuo, the mixture was diluted with water to 8 mL, extracted 4 times with ethyl acetate (6 mL), then the aqueous layer was neutralized with silver carbonate and concentrated after filtration. Direct co-thin-layer chromatography (chloroform–MeOH–H₂O, 6:4:1) comparison with reference d-glucose and positive optical rotation established that the glucose in compound 1 was in d-configuration (Rf = 0.13).¹¹

Cytotoxicity

An MTT assay was used to measure the in vitro cytotoxicity of isolated compounds. The MCF-7 was cultured in 96-well plates for 24 hours and then treated with test compounds at various concentrations (3-400 μM) for 72 hours. After treated with MTT solution (0.5 mg/mL in phosphate-buffered saline) for another 4 hours, the medium was discarded, 100 µL of dimethylsulfoxide was added to dissolve the produced formazan. The absorbance was measured at 490 nm using a microplate reader. Doxorubicin was used as a positive control. Each experiment was carried out in triplicate. The IC₅₀ values were calculated using Graphpad Prism software.

Among the isolated 7 compounds, isocorilagin shows moderate cytotoxicity with an IC₅₀ value of 41.31 µM while isomallotusinin which has a similar structure gives an IC₅₀ value higher than 200 uM. Compound 1 shows no cytotoxicity to MCF-7 cell lines.

Supplemental Material

Supplemental Material 1 - Supplemental material for Phytochemical Investigation of Erodium oxyrrhynchum M. Bieb

Supplemental material, Supplemental Material 1, for Phytochemical Investigation of Erodium oxyrrhynchum M. Bieb by Lanyan Jin, George Fayvush, Manana Khutsishvili, Daniel Atha and Robert P. Borris in Natural Product Communications

Footnotes

Declaration of Conflicting Interests

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

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this work was financially supported by the National Basic Research Program (973) of China (2015CB856500).

ORCID iD

Robert P. Borris

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Supplementary Material

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