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Phenanthrene derivatives from roots and rhizomes of Asarum heterotropoides var. mandshuricum
Accepted Manuscript Phenanthrene derivatives from roots and rhizomes of Asarum heterotropoides var. mandshuricum
Yu Jing, Yi-Fan Zhang, Ming-Ying Shang, Jie Yu, Jia-Wei Tang, Guang-Xue Liu, Yao-Li Li, Xiao-Mei Li, Xuan Wang, Shao-Qing Cai PII: DOI: Reference:
S0367-326X(16)30615-3 doi: 10.1016/j.fitote.2017.01.008 FITOTE 3557
To appear in:
Fitoterapia
Received date: Revised date: Accepted date:
21 October 2016 16 January 2017 21 January 2017
Please cite this article as: Yu Jing, Yi-Fan Zhang, Ming-Ying Shang, Jie Yu, Jia-Wei Tang, Guang-Xue Liu, Yao-Li Li, Xiao-Mei Li, Xuan Wang, Shao-Qing Cai , Phenanthrene derivatives from roots and rhizomes of Asarum heterotropoides var. mandshuricum. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Fitote(2017), doi: 10.1016/j.fitote.2017.01.008
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Phenanthrene Derivatives from Roots and Rhizomes of Asarum heterotropoides var. mandshuricum
The State Key Laboratory of Natural and Biomimetic Drugs,
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a
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Xuan Wang a, Shao-Qing Cai a, **
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Yu Jing a, c, Yi-Fan Zhang a, Ming-Ying Shang a, *, Jie Yu a, Jia-Wei Tang b, Guang-Xue Liu a, Yao-Li Lia, Xiao-Mei Li b,
Peking University Health Science Center, No.38, Xueyuan Road, Beijing 100191, China
Renal Division, Department of Medicine, First Hospital, and Institute of Nephrologist, Peking University, The Key
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b
Tonghua Gold-horse Pharmaceutical Group Co., Ltd., Beijing 100025, China
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C
China
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Laboratory of Renal Disease, The Ministry of Health of China, Beijing 100034 ,
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* Corresponding author. Tel./Fax: +86 10 8280 2534.
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** Corresponding author. Tel./Fax: +86 10 8280 1693.
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E-mail address: [email protected] (M,-Y. Shang), [email protected] (S, -Q. Cai).
Abstract Five new phenanthrene derivatives: 9-ethoxy-7-methoxy-aristololactam IV (1), norcepharadione A N-β-D-glucopyranoside (2), aristololactamoside I (3), aristololactamoside II (4) and aristothiolactoside (5) together with eleven known phenanthrene derivatives (6—16) were isolated from the ethanol extract of the roots and rhizomes of Asarum heterotropoides var. mandshuricum. The aristololactams with substitution of ethoxy at C-9 position (1, 9, and 10) and the sulfur-containing phenanthrene derivative (5) were reported in the genus Asarum for the first time. Furthermore，six
phenanthrene glucoside derivatives (2—5, 13 and 14) -1-
ACCEPTED MANUSCRIPT were also found in this genus for the first time and compounds 7 and 9—15 were isolated from the genus Asarum for the first time. Six of them (1, 2, 9, 10, 13 and 14) were submitted to cytotoxicity test against human renal proximal tubular epithelial cell lines (HK-2) using MTT and LDH assays. Compounds 1 and 10 showed significant cytotoxic activity against HK-2 cell lines with IC50 values of 18.18 and 20.44 μmol/L in MTT assay and 84.36 and 35.06 μmol/L in LDH assay, respectively. Compound 9 showed moderate cytotoxicity in MTT assay with IC50 values of 95.60 μmol/L, but no cytotoxicity in LDH assay. Compounds 2, 13 and 14 showed cytotoxic effect in
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neither MTT assay nor LDH assay. Considering the other nephrotoxic phenanthrene derivatives (6, 8, 12, 15 and 16) previously tested, the results implied the potency of renal toxicity of this herb used as a medicine.
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Keywords: Asarum heterotropoides var. mandshuricum; Phenanthrene derivatives; Aristolochic acid;
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Aristololactam; Nephrotoxicity;
Chemical compounds studied in this article
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Aristololactam I (PubChem CID: 96710); Aristololactam IV (PubChem CID: 5319404); 9-ethoxy-aristololactam
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I (PubChem CID: 195368); Aristolochic Acid IVa (PubChem CID: 161218); Cepharadione A (PubChem CID: 94577 ); Aristolochic acid I (PubChem CID: 2236)
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1. Introduction
Three classes of phenanthrene derivatives, namely aristolochic acids, aristolactams and 4, 5-dioxoaporphines,
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are a small group of compounds primarily found in Aristolochiaceae [1]. Up to now, aristolochic acids mainly
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occur in the genus Aristolochia and Asarum [2], while aristololactams and 4, 5-dioxoaprophine derivatives are widely found in Annonaceae [1, 2], Saururaceae [3], Menispermaceae [4] andPiperaceae [5]. Aristolochic acids and aristololactams were shown to possess immunostimulatory and anti-inflammatory as well as nephrotoxic, carcinogenic, and mutagenic activities [1]. But they are paid most close attention in recent years among known phenanthrene derivatives for the reason that some of them are associated with a high risk of nephrotoxicity and upper urinary tract carcinoma (UUC), which lead to cause Aristolochic Acid Nephrophathy (AAN) [6–8]. As previously reported，aristolochic acid I is responsible for the destructive fibrotic process in the kidney, which leads to permanent kidney damage in a short period because of aristolochic acid derived-DNA adducts [9].
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ACCEPTED MANUSCRIPT Moreover, aristololactam I was also proved to have cytotoxicity against human proximal tubular cells (HK-2) in recent researches of our group [10, 11]. The genus Asarum contains about 70 species growing in both temperate and tropical regions, and a number of Asarum species are used in herbal medicines throughout the world. Up to date, plants belonging to the genus Asarum were reported to contain a few of phenanthrene derivatives, including aristololactam I [12–17], 5-methoxyaristololactam I [16], 9-hydroxyaristololactam I [15], aristololactam Ia [13], aristololactam II [13],
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aristololactam AII [13, 18], aristololactam III [18], 7-methoxy-aristololactam IV [12–15, 17, 19, 20],
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aristololactam VII [21], aristolochic acid I [12, 13, 15, 16, 22], 7-hydroxyaristolochic acid I [15, 16], aristolochic acid Ia [15], aristolochic acid IV [15], aritolochic acid IVa [14, 17], 4-demethoxyaristolochic acid BII [15], aristololide [13]，aristolic acid I [13], aristolic acid II [15]. The amount of phenanthrene derivatives of the genus
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Asarum is significantly less than that found in the genus Aristolochia [1, 2].
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The root and rhizome of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag is one of the most important crude drug of the genus Asarum in traditional Chinese medicine, which is described as Asari
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Radix et Rhizoma (Xixin) in the Chinese Pharmacopoeia (2010 edition) used as an anodyne, antitussive and
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anti-allergic remedy. It is widely used to treat various diseases such as aphthous stomatitis, headache, toothache and inflammatory in China, Korea and Japan [23]. Although a number of constituents have been isolated, such as
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lignans [24, 25], amides [26], terpenes [27] and flavones [28], only three phenanthrene derivatives including aristololactam I，7-methoxy-aristololactam I and aristolochic acid IVa were isolated from this crude drug [14, 17].
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And a few papers reported that trace amounts (42.2 μg/g, 16.8μg/g, 26.47μg/g) of aristolochic acid I could be
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detected in this crude drug [29–31].
In this paper we focused our interest on phenanthrene derivatives of this crude drug and nephrotoxicity of this kind of compounds. As a result, sixteen phenanthrene derivatives (1—16) containing eleven aristololactams, two aristolochic acids, two 4, 5-dioxoaporphines and one sulfur-containing phenanthrene derivative were isolated and identified from this crude drug. In order to evaluate nephrotoxicity of them, six constituents (1, 2, 9, 10, 13 and 14) were submitted to cytotoxicity test against HK-2 cells using MTT and LDH assays. With the aim of replenishing previous researches on phenanthrene derivatives from the genus Asarum, we reported the isolation, as well as the structure elucidation and nephrotoxicity evaluation of phenanthrene derivatives from Asarum
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ACCEPTED MANUSCRIPT heterotropoides var. mandshuricum. The results can provide us an important clue whether this crude drug can be applied safely in clinic or not. 2. Materials and methods 2.1. General experimental procedures Melting points were obtained on an XT-4A micromelting point apparatus without correction. Optical rotations were determined on a Perkin-Elmer 243B digital polarimeter. UV spectra were carried out on a Cary 300 UV-Vis
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spectrophotometer. A Nicolet NEXUS-470 FTIR spectrophotometer was used for scanning IR spectroscopy.
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NMR spectra were recorded on Varian INOVA-500. The chemical shifts are expressed as δ (ppm) values using solvent as an internal standard, and coupling constant, J, are in Hz. Mass spectra were detected with Bruker APEX IV FT and ABI Q-STAR mass spectrometers. GC was carried out on a Shimadzu GC-2010 series system
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fitted with a FID detector and performed with a DB-1701 column (30 m × 0.25 mm i.d., 0.25 μm film thickness).
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Semi preparative HPLC was conducted on an Alltima C18 column (10 mm i.d. × 250 mm, 10 μm) equipped with an Alltech 426 HPLC pump and an Alltech UVIS 2000 detector. Column chromatography were performed with
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Silica gel (200—300 mesh, Qingdao Marine Chemical Co., Ltd.), Sephadex LH-20 gel (Pharmacia Co., Ltd.). TLC analysis was performed on silica gel (400 mesh, Qingdao Marine Chemical Co., Ltd.) and precoated
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polyamide plates (0.2 mm, Zhejiang Siqing biochem Co., Ltd.) plates. D-glucose was obtained from Beijing
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Chemical Reagent Company. All other chemical solvents used for isolation were of analytical grade (Beijing Beihua Fine Chemicals Co., Ltd.). Fractions were monitored by TLC and spots were detected by UV
2.2. Plant material
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illumination.
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The roots and rhizomes of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. were purchased from Benxi city, Liaoning province, China, in May 2005, and authenticated by Professor Shao-Qing Cai of Peking University Health Science Center. Voucher specimen (No. 5066) was deposited in the Herbarium of Pharmacognosy, School of Pharmaceutical Sciences, Peking University, Beijing, China. 2.3. Extraction and isolation The air-dried and powdered roots and rhizomes of A. heterotropoides var. mandshuricum (36 kg) were extracted three times (2 h, 1.5 h, 1.5 h for each) under reflux with 95% ethanol and then three times (2 h, 1.5 h, 1.5 h for each) with 50% ethanol successively. The
combined extracts were concentrated under reduced -4-
ACCEPTED MANUSCRIPT pressure to give a dark brown extract (9.2 kg), then 7.8 kg of it was suspended in H2O (16.5 L) and partitioned sequentially with petroleum ether (60—90 ℃) (4 × 5 L), CHCl3 (4 × 5 L), EtOAc (4 × 5 L), and n-BuOH (saturated with water) (4 × 5 L), respectively. The CHCl3 layer (231 g) was fractionated on silica gel column chromatography eluting with a gradient of petroleum ether—EtOAc (10:1 to 0:1) to obtain sixteen fractions C1—C16. Fraction C10 and C11 were left to stand overnight and yellow powders as precipitate were collected respectively, then each yellow powder was purified by Sephadex LH-20 eluting with CHCl3—MeOH (6:4) to
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afford compounds 6 (amount 57.8 mg, yield 2.19 μg/g) and 7 (amount 28.5 mg, yield 1.08 μg/g). The residue of
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fraction C11 was subjected to silica gel column chromatography eluting with a gradient of CHCl3—MeOH (1:30 to 1:35) to yield four subfractions C11-1—C11-4. C11-1 yielded a yellow powder, collected by filtering, which showed two high yellow fluorescence spots on TLC under UV illumination. Then the yellow powder was
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separated by semi-preparative HPLC eluting with a gradient of MeOH—H2O to yield compounds 8 (amount 10.2
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mg, yield 0.39 μg/g) and 10 (amount 8.7 mg, yield 0.33 μg/g). The residue of C11-1 was then subjected to chromatography on silica gel eluting with petrol—Me2CO to yield seven tertiary fractions (C11-1-1—C11-1-7).
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C11-1-1 was further separated and purified by Sephadex LH-20 eluting with CHCl3—MeOH (1:1) and recrystallized from MeOH to afford compounds 1 (amount 6.1 mg, yield 0.23 μg/g) and 9 (amount 5.1 mg, yield
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0.19 μg/g); Fraction C12 was chromatographed on silica gel with a gradient of CHCl3－MeOH to give four
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subfractions C12-1－C12-4. C12-3 was further divided into five subfractions C12-3-1－C12-3-5 with repeated
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silica gel column chromatography eluting with CHCl3－MeOH (80:1 to 15:1). C12-3-3 was subsequently further separated on silica gel column with CHCl3—EtOAc and purified by semi-preparative HPLC using a gradient of
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MeOH—H2O as eluent to afford compound 12 (amount 4.2 mg, yield 0.16 μg/g). Fraction C13 was chromatographed on a column packed with silica gel and eluted with CHCl3—MeOH, and subsequently purified by recrystallization from MeOH to afford compound 15 (amount 3.6 mg, yield 0.14 μg/g). The EtOAc layer (50 g) was fractionated on silica gel CC eluting with a gradient of CHCl3－MeOH (10:1 to 0:1) to yield ten fractions E1—E10. Fraction E2 gave yellow powder, which washed with MeOH to afford compound 11 (amount 120 mg, yield 4.37 μg/g). Fraction E3 gave yellow powder which was further separated and purified by semi-preparative HPLC with MeOH—H2O as eluent to yield compound 16 (amount 2.3 mg, yield 0.08 μg/g). Fraction E6 yielded yellowish green flaky crystal, which was further washed with MeOH to afford compound 13 (amount 160 mg, -5-
ACCEPTED MANUSCRIPT yield 5.82 μg/g); Fraction E8 was separated on silica gel column chromatography eluting with CHCl3－MeOH－ H2O (8:1:0.1, 5:1:0.1) and then purified by semi-preparative HPLC with MeOH－H2O as eluent to yield compounds 14 (amount 7.0 mg, yield 0.25 μg/g) and 4 (amount 2.3 mg, yield 0.08 μg/g). The n-BuOH layer (400 g) was fractionated on silica gel column chromatography eluting with a gradient of CHCl3－MeOH (7:1 to 0:1) to yield fourteen fractions B1—B14. Fraction B6 was further separated on silica gel CC eluting with CHCl3－
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MeOH－H2O (5:1:0.1 to 2:1:0.1) to give nine subfractions B6-1－B6-9. B6-4 was subjected to repeated silica gel
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column chromatography eluting with CHCl3－MeOH－H2O (6:1:0.1), followed by semi-preparative HPLC with a gradient of MeOH－H2O as eluent to afford compounds 2 (amount 5.6 mg, yield 0.21 μg/g) , 3 (amount 2.1 mg, yield 0.08 μg/g) and 5 (amount 3.5mg, yield 0.13 μg/g).
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9-ethoxy-7-methoxy-aristololactam IV (1): yellow needles (MeOH); mp 248—249 ℃; UV (MeOH) λmax 238, 288, 299, 338, 403 nm; IR (KBr) νmax 3432—3165, 1690, 1604, 1506, 1473, 1406, 1357, 1305, 1271, 1163, 1130,
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1049, 949 cm-1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) spectroscopic data are
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shown in Table 1; EIMS m/z 397 [M]+ (100), 368 (41), 353 (31), 338 (22), 325 (19), 310 (24), 282 (13), 268 (17), 239 (13), 184 (18); HRESIMS m/z 398.1230 [M + H]+ (calcd. for C21H20NO7, 398.1234).
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Norcepharadione A N-β-D-glucopyranoside (2): red amorphous powder; [α]20D —15.7 (c 1.40, DMSO); UV
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(MeOH) λmax 217, 235, 279, 302, 314, 436 nm; IR (KBr) νmax 3447, 3421, 1647, 1595, 1416, 1365, 1041 cm-1; 1H NMR (DMSO-d6, 500 MHz) δ 8.97 (1H, m, H-11), 8.42 (1H, s, H-7), 8.02 (1H, s, H-3), 8.00 (1H, m, H-8), 7.74
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(2H, m, H-10, 9), 6.60 (2H, s, -OCH2O-), 6.26 (1H, d, J = 10.0 Hz, H-1′), 4.07—3.36 (6H, m, H-4′, 6′, 2′, 3′, 5′); C NMR (DMSO-d6, 125 MHz) δ 173.6 (C-4, C=O), 156.8 (C-5, C=O), 151.4 (C-1), 147.4 (C-2), 131.0 (C-7a),
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129.2 (C-8), 129.1 (C-6a), 128.2 (C-10), 127.6 (C-9), 125.9 (C-11), 124.6 (C-11a), 122.5 (C-3a), 121.4 (C-11c), 117.5 (C-7), 114.2 (C-11b), 107.7 (C-3), 103.7 (-OCH2O-), 85.0 (C-1′), 80.5 (C-5′), 77.5 (C-3′), 69.3 (C-2′), 68.8 (C-4′), 60.6 (C-6′); ESIMS m/z 454 [M + H]+, 476 [M + Na]+; HRESIMS m/z 476.0967 [M + Na]+ (calcd. for C23H19NO9Na, 476.0952). Aristololactamoside I (3): yellow amorphous powder; [α]20D —66.6 (c 0.30, DMSO); UV (MeOH) λmax 210, 240, 263, 285, 329, 349, 404 nm; IR (KBr) νmax 3415, 1673, 1627, 1466, 1406, 1304, 1046, 612 cm-1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) spectroscopic data are shown in Table 1; ESIMS m/z 634 [M + H]+, 656 [M + Na]+; HRESIMS m/z
634.1771 [M + H]+ (calcd for C29H32NO15, 634.1766). -6-
ACCEPTED MANUSCRIPT Aristololactamoside II (4): yellow amorphous powder; [α]20D —28.3 (c 0.46, DMSO); UV (MeOH) λmax 212, 256, 286, 326, 383 nm; IR (KBr) νmax 3393, 1663, 1545, 1467, 1416, 1374, 1288, 1258, 1030, 628 cm-1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) spectroscopic data are shown in Table 1; ESIMS m/z 444 [M + H]+, 466 [M + Na]+, 909 [2M + Na]+; HRESIMS m/z 444.1281 [M + H]+ (calcd for C22H22NO9, 444.1289). Aristothiolactoside (5): yellow amorphous powder; [α]20D —30.0 (c 0.70, DMSO); UV (MeOH) λmax 251, 272,
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294, 310, 341, 361, 416; nm; IR (KBr) νmax 3420, 2920, 1663, 1613, 1461, 1424, 1357, 1275, 1077, 1047, 638
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cm-1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) spectroscopic data are shown in Table 1; ESIMS m/z 651 [M + H]+, 673 [M + Na]+; HRESIMS m/z 651.1399 [M + H]+ (calcd for C29H31SO15,
2.4 Acid Hydrolysis of Compound 2－5, and GC Analysis
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651.1378).
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Compound 2－5 (each about 1 mg) in 2 N HCl (3 ml) were refluxed at 85 ℃ for 3 h, respectively. After cooling, the reaction mixture was neutralized with NaHCO3 and successively extracted with EtOAc (4 × 3 ml). The
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aqueous layer was then evaporated to dryness and the residue was dissolved in anhydrous pyridine (200 μl), and
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L-cysteine methyl ester hydrochloride (0.06 mol/L, 200 μl) was added. The mixture was stirred at 60 ℃ for 1 h.
Then 150 μl trimethylsilylation reagent hexamethyldisilazane—trimethyl chlorosilane (HMDS—TMCS, 3:1) was
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added, and the mixture was stirred at 60 ℃ for an additional 30 min. After centrifugation, the supernatant was concentrated under a stream of N2. The residue was portioned between n-hexane and H2O (0.2 ml each), and the
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n-hexane layer (2 μl) was analyzed by GC for sugar identification (detector, FID; injection temperature, 260 ℃;
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detector temperature, 280 ℃; temperature gradient system for the oven, 160 ℃ for 1 min and then raised to 230 ℃ at rate of 5 ℃/min; carrier gas, N2; flow rate, 1 mL/min). D-glucose were identified for compounds 2－5 by comparion with retention time of authentic D-glucose (tR = 20.76 min) after treatment in the same manner. 2.5 Cytotoxicity Assay in HK-2 Cell Lines. Cytotoxicity was assessed by two methods as described previously [32], including LDH leakage into culture medium, which indicates the integrity of cellular membrane, and MTT conversion into insoluble formazan stain, which shows functional mitochondrial dehydrogenase. All experiments were performed with HK-2 cells, which were obtained from American Tissue Culture Collection (Rockville, Md., USA). All tested compounds were -7-
ACCEPTED MANUSCRIPT dissolved in dimethylsulfoxide (DMSO) (Sigma), and then diluted in Dulbeccos′s modified Eagle′s medium (DEME) (Gibco Invitrogen Corp.) to the indicated concentration of experiments. Results and Discussion The EtOH extract of the roots and rhizomes of Asarum heterotropoides var. mandshuricum was suspended in H2O and partitioned with petroleum ether, CHCl3, EtOAc and n-BuOH, successively. The CHCl3, EtOAc and n-BuOH extracts were separated and purified by various column chromatographic methods to obtain sixteen
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phenanthrene derivatives, including five new compounds (1—5) and eleven known compounds (6—16) (Fig. 1).
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Their structures were elucidated based on the spectroscopic methods. The eleven known compounds were identified as aristololactam I (6) [33], aristololactam IV (7) [34], 7-methoxy-aristololactam IV (8) [33], 9-ethoxy-aristololactam I (9) [35], 9-ethoxy-aristololactam IV (10) [36], aristolochic acid IVa (11) [37],
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aristololactam IVa (12) [38], aristololactam II N-β-D-glucopyranoside (13) [39], aristololactam Ia
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N-β-D-glucopyranoside (14) [40], cepharadione A (15) [41] and aristolochic acid I (16) [33] by comparison of the spectroscopic data with those reported in the literatures. Moreover, compounds 7 and 9—15 were isolated from
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the genus Asarum for the first time.
Compound 1 was obtained as a yellow needle. The molecular formula was deduced as C21H19NO7 by its
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HRESIMS (m/z 398.1230 [M + H]+, calcd. 398.1234). Its UV spectrum in methanol showed characteristics of a
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phenanthrene derivative at 238, 288, 299, 338 and 403 nm [42]. The IR spectrum showed absorptions for imino group (3432—3165 cm-1) and lactamic carbonyl group (1690 cm-1). The 1H NMR spectrum of 1 showed the
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presence of three methoxyls δ 3.94 (3H, s), 3.87 (3H, s) and 3.83 (3H, s). The two singlet signals of aromatic protons at δ 7.96 (1H, s) and 7.57 (1H, s) assignable to H-5 and H-2, respectively, which was confirmed by the
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correlation between H-5 and C-7, C-4a, and C-8a, H-2 and C-10a, C-4, and carbonyl carbon in HMBC experiment. The methylenedioxy group was observed as a singlet at δ 6.43 (2H, s). The other singlet at δ 10.91 (1H, s) was assigned to NH. The 1H NMR spectrum showed the presence of an oxygenated methylene signal at δ 4.03 (2H, q, J = 7.0 Hz) and a methyl signal at δ 1.40 (3H, t, J = 7.0 Hz), which were attributed to an ethoxyl group. This was further supported by the correlations of the protons of above two groups in 1H-1H COSY spectrum. The HMBC experiment showed the correlation between the protons of the oxygenated methylene group at δ 4.03 and C-9 (Fig. 2), so the ethoxyl group should be located at C-9. The 1H, 13C NMR and DEPT
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ACCEPTED MANUSCRIPT spectra showed the expected one methyl, two methines, thirteen quaternary carbons, three methoxyl groups, one methylenedioxy group, and one oxygenated methylene group. According to 1H and 13C NMR spectra, the only difference between compound 1 and 9-ethoxy-aristololactam IV (10) [36] was the presence of one methoxyl at δ 3.94 (3H, s) attached to C-7. The full assignments of 1H and 13C NMR data of compound 1 was achieved by comparing them with those of 9-ethoxy-aristololactam I (9) [35] and 9-ethoxy-aristololactam IV (10), which
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compound 1 was established as 9-ethoxy-7-methoxy-aristololactam IV.
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further assigned by using an HMBC experiment (Fig. 2). On the basis of the above spectroscopic analysis,
Compound 2 was obtained as a red amorphous powder. The molecular formula of compound 2 was established to be C23H19NO9 by HRESIMS (m/z 476.0967 [M + Na]+, calcd. 476.0952). The UV spectrum showed absorptions
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of a 4, 5-dioxoaporphine derivative at 217, 235, 279, 302, 314 and 436 nm [43]. The presence of hydroxyl and carbonyl groups were supported by the IR spectrum peaks at 3447—3421 and 1647 cm-1. The 1H NMR spectrum
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didn’t show the presence of methoxyl and NH groups. In the 1H NMR spectrum, one set of four mutually coupled proton signals at δ 8.97 (1H, m), 8.00 (1H, m) and 7.74 (2H, m) corresponded to H-11, H-8, H-10 and H-9,
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respectively. The singlet signals at δ 8.02 (1H, s) and 8.42 (1H, s) were assigned to H-3 and H-7, respectively,
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which were inferred by the HMBC correlations between H-3 and C-1, C-11c, and H-7 and C-11a, C-11c (Fig. 2). The singlet signal at δ 6.60 (2H, s) was assigned to a methylenedioxy, because the protons showed the long-range
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correlations to C-1 and C-2 in HMBC spectrum. In the 1H NMR spectrum of compound 2 a doublet signal at δ
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6.26(1H, d, J = 10.0 Hz) was assigned as an anomeric proton of glucose moiety. The 1H and 13C NMR data as well as acid hydrolysis and GC comparison with an authentic sample indicated the presence of a moiety. The β-configuration of the glycoside linkage was inferred by its large coupling
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D-glucopyranosyl
constant J = 10.0 Hz of the anomeric proton at δ 6.26 (1H, d). Comparing with N-methoxy norcepharadione A [44], the absence of a methoxyl group in 2 suggested that the glucosyl moiety likely attached to the nitrogen of the aglycone. To confirm the connection between the glucosyl and the phenanthrene unit of compound 2, a HMBC experiment was conducted. The results showed the correlations between the anomeric proton H-1′ and C-6a, C-5 (Fig. 2), which further supported the above conclusion. Therefore, the structure of compound 2 was established to be norcepharadione A N-β-D-glucopyranoside. Compound 3 was obtained as a yellow amorphous
powder. The molecular formula was suggested to be -9-
ACCEPTED MANUSCRIPT C29H31NO15 by HRESIMS (m/z 634.1771 [M + H]+, calcd. 634.1766). The UV spectrum exhibited the characteristic at 240, 263, 285, 329, 349 and 404 nm of a phenanthrene chromophore [42]. The IR bands at 3415 and 1673 cm-1 revealed the presence of hydroxyl and lactamic carbonyl group. The signal for H-2 was observed at δ 7.66, which confirmed by the correlations between H-2 and C-10a, C-4, and carbonyl group in HMBC spectrum (Fig. 2). The aromatic region of the 1H NMR spectrum contained an ABC pattern signals at δ 8.30(1H, d, J = 8.0 Hz), 7.58(1H, t, J = 8.0 Hz) and 7.30(1H, d, J = 8.0 Hz), which was ascribed to H-5, H-6 and H-7, respectively.
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One methoxyl group proposed to be attached to C-8 was further confirmed by HMBC correlations between H-6,
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methoxyl protons (δ 3.95) and C-8. The two singlets at δ 9.88 (1H, s) and 6.46 (2H, s) were attributed to NH and methylenedioxy, respectively. The 1H and 13C NMR data suggested compound 3 was a monomethoxyl aristololactam with 3, 4-methylenedioxy substituent. The 13C NMR singals at δ 104.6, 103.0, 76.6, 76.4, 75.9,
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75.8, 73.9, 73.0, 69.8, 69.7, 68.4 and 60.8 indicated the presence of two glucopyranose moieties [45]. The typical coupling constants of two anomeric protons at δ 4.92 (1H, d, J = 7.5 Hz) and 4.09 (1H, d, J = 7.5 Hz) suggested
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the β-conformations for two glucoses. The acid hydrolysis and GC comparison with an authentic sample
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indicated the presence of a D-glucose. According to above analysis, C-9 is a suitable position available for the glycosidic linkage, which was further supported by the correlation between the anomeric proton H-1′ and C-9 in
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HMBC spectrum (Fig. 2). Furthermore, in 13C NMR spectrum the C-6′ signal in one glucosyl downfield shifted to
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δ 68.4, which indicated that the second anomeric carbon C-1′′ should be linked to C-6′. Moreover, the correlation between H-1′′ and C-6′ in HMBC spectrum also proved the above conclusion (Fig. 2). The combined evidence
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supported the structure of compound 3 for aristololactam I
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9-O-β-D-glucopyrannosyl(1→6)-O-β-D-glucopyranoside, and it was named as aristololactamoside I. Compound 4 was obtained as a yellow amorphous powder. Its molecular formula was established as C22H21NO9 by HRESIMS (m/z 444.1281 [M + H]+, calcd. 444.1289). The UV spectrum of compound 4 showed absorptions at 212, 256, 286, 326 and 383 nm, which corresponded to the characteristic of a phenanthrene chromophore [42]. The IR spectrum indicated the presence of hydroxyl or imino group (3493 cm-1) and lactamic carbonyl group (1663 cm-1). The 1H NMR spectrum revealed the presence of an imino proton at δ 10.59 (1H, s). Through analysis of 1H NMR and 1H-1H COSY spectra, an ABC system aromatic protons signals were observed at δ 8.99 (1H, brs), 7.49 (1H, t, J = 8.0 Hz) and 7.16 (1H, d, J = 8.0Hz), which were assigned to H-5, H-6 and H-7, - 10 -
ACCEPTED MANUSCRIPT respectively. And the correlations between H-7 and C-5, C-8a, H-6 and C-4b in HMBC spectrum further confirmed above conclusions (Fig. 2). The other two singlets at δ 7.85 (1H, s) and δ 7.46 (1H, s) were attributed to H-2 and H-9, respectively, which were supported by the cross peaks between H-2 and C-10a, H-9 and C-4b, C-10a in HMBC spectrum (Fig. 2). The 1H NMR spectrum also showed methoxyl protons signal at δ 3.99 (3H, s). And the correlations between H-9, the methoxyl protons (δ 3.99) and C-8 in HMBC spectrum inferred that the methoxyl attached to C-8. In addition, the 1H and 13C NMR spectra of compound 4 exhibited the existence of
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β-glucopyranose moiety, which supported by coupling constants of the anomeric proton at δ 4.82(1H, d, J = 8.0
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Hz). And acid hydrolysis confirmed the absolute configuration of the sugar moieties as D-glucose as determined by GC analysis. Comparing the 1H and 13C NMR data of compound 4 with those of enterocarpam II [46], we concluded that the glucosyl moiety most likely attached to C-3 (δ 146.1) or C-4 (δ 141.2). In the NOESY
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experiment, there was no NOE correlation between H-2 and the anomeric proton (H-1′). On the other hand, the
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signal for C-4 of compound 4 at δ 141.25 was much more high field than the corresponding carbon signal at δ 145.75 of 10-amino-3, 4-dihydroxyl phenanthrene-1-carboxylic acid lactam [47] due to glycosidation effect of
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phenolic hydroxyl at C-4. Based on the above evidence, we confirmed that the glycosidic linkage of compound 4 was determined to be at C-4. But the HMBC correlation between the anomeric proton and C-4 (δ 141.2) was not
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observered due to the small quality of compound 4. In combination with 1H, 13C NMR and HRESIMS data, it
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could be concluded that a hydroxyl group existed in the structure and attached to C-3. On the basis of the above spectroscopic data, the structure of compound 4 was determined to be enterocarpam II 4-O-β-D-glucopyranoside
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and named as aristololactamoside II.
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Compound 5 was obtained as yellow amorphous powder. The HRESIMS, exhibiting a molecular ion peak at m/z 651.1399 [M + H]+, indicated that the molecular formula was C29H30SO15 (calcd. 651.1378). The UV spectrum exhibited absorption at 251, 272, 294, and 341 nm, which correspongding to a phenanthrene chromophore. The IR spectrum showed absorptions for hydroxyl
(3420 cm-1 )and carbonyl (1663 cm-1) groups,
respectively. Its NMR spectra exhibited the presence of one methoxyl group [δH 4.01 (3H, s) and δC 56.3], one methylenedioxy group [δH 6.55 (2H, d, J = 23.5 Hz) and δH 104.5], two aromatic protons [δH 7.75 (1H, s) and δC 105.6; δH 8.14 (1H, s) and δC 117.2], two meta-coupled aromatic protons [δH 8.01 (1H, d, J = 2.0 Hz) and δC 104.9; δH 7.06 (1H, d, J = 2.0 Hz) and δC 100.0]. Moreover, the 1H NMR spectrum also showed the presence of - 11 -
ACCEPTED MANUSCRIPT two anomeric protons of glucose moiety at δ 5.17 (1H, d, J = 7.5 Hz, H-1′) and δ 4.40 (1H, d, J = 7.5 Hz, H-1′′), together with two sets of protons signal of glucose moiety including seven hydroxyl groups [δ 5.51 (1H, brs), δ 5.09 (1H, d, J = 3.0 Hz), δ 5.02 (1H, d, J = 4.5 Hz), δ 4.99 (1H, d, J = 5.5 Hz), δ 4.82 (1H, d, J = 2.5 Hz), δ 4.70 (1H, t, J = 5.5 Hz) and δ 4.61 (1H, t, J = 5.5 Hz)] and twelve glucosyl protons [δ 3.71—3.79 (2H, m), δ 3.52—3.58 (4H, m), δ 3.39—3.44 (2H, m), δ 3.18—3.24 (2H, m) and δ 3.06—3.12 (2H, m)]. The 13C-NMR and HMQC spectra showed signals for fourteen aromatic carbons, one carbonyl group (δC 190.1), one methylenedioxy
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group (δC 104.5), one methoxyl group (δC 56.3) and twelve glucosyl carbons (δC 104.5, 100.5, 87.8, 77.1,
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77.1,76.3, 74.0, 72.1 70.3, 68.3 61.3 and 60.6). The HRESIMS indicated compound 5 was a sulfur-containing compound. So the carbonyl group (δC 190.1) was lower field than the lactam carbonyl group of aristololactam derivatives due to the deshielding effect of sulfur atom. The 1H and 13C NMR spectroscopic data of compound 5
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were very similar to that reported in the literature for aristothiolide [36], except for the absence of a methoxyl group at C-6. The NMR spectra showed the presence of two glucopyranosyl moieties in compound 5. Acid
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hydrolysis of compound 5 liberated D-glucose, which was determined by GC analysis. And the β-configurations
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of two glucose units were determined from the coupling constant of the two anomeric protons. The location of the inner glucose moiety at C-6 in compound 5 was corroborated by HMBC experiment (Fig. 2) that showed
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correlation between the anomeric proton (δ 5.17, H-1′) and C-6 (δ 157.7). In addition, the connectivities of two
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β-D-glucopyranosyl moieties in compound 5 were clarified by HMBC experiment, in which long-range correlation were observed between anomeric proton (δH 4.40, H-1′′) of the terminal glucose moiety and C-3′ (δ
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87.8) of the inner glucose moiety (Fig. 2). Additionally, the HMBC correlations (Fig. 2) of the aromatic protons H-2 (δH 7.75) with C-4 (δC 148.9), C-10a (δC 132.7) and carbonyl carbon (δC 190.1), H-9 (δH 8.14) with C-3 (δC
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150.4), C-4b (δC 127.7) and C-10a (δC 132.7), H-5 (δH 8.01) with C-4a (δC 120.0), C-8a (δC 118.3) and C-7 (δC 100.0), H-7 (δH 7.06) with C-5(δC 104.9) and C-8a (δC 118.3) as well as the methylenedioxy protons (δH 6.55) with C-3 (δC 150.4) and C-4 (δC 148.9) were observed. On the basis of the above results and spectral data, the structure of compound 5 was recognized as 6-demethy aristothiolide 6-O-β-D-glucopyrannosyl(1→3)-O-β-D-glucopyranoside and named as aristothiolactoside. Compounds 1 as well as the known 9 and 10 all have an ethoxyl group at C-9 position in their structures, which reported in the Asarum plants for the first time. This kind of phenanthrene derivative with an ethoxyl group is
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ACCEPTED MANUSCRIPT extremely rare in nature, for only compounds 9 and 10 had been obtained only from Aristolochia mollisimma Hance. [35] and Aristolochia argentina [36], respectively. There were just sixteen phenanthrene glycosidic derivatives found in the genus Aristolochia of Aristolochiaceae so far, including fourteen monoglycosides and two single-chain diglucosides [1, 2]. However, until now no phenanthrene glycosidic derivative was obtained from the genus Asarum. In this paper we first reported six phenanthrene glycosidic derivatives (2—5, 13 and 14) isolated from Asarum heterotropoides var. mandshuricum of the genus Asarum. Moreover, compound 3 and 5
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time that phenanthrene diglycosidic derivatives were isolated from this genus.
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exhibited a single-chain glycoside consisting of two glucoses in their structures, respectively, which was the first
Compound 4 was isolated as an oxygen glycoside with glycosidic linkage and a hydroxyl group at the C-4 and C-3 position, respectively. To our best knowledge, aristololactams having a methoxyl and a hydroxyl group at
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C-3(4) and C-4(3) positions, two methoxyl groups or methylenedioxy group at C-3 and C-4 positions, are
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common in the plants of Aristolochiaceae [1, 2]. But hitherto there have been no report of aristololactam like compound 4 with the glycosidic linkage at C-4 position having been isolated from nature. In addition, compound
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5 was identified as a rare sulfur-containing phenanthrene glycosidic derivative with aristothiolide as aglycone, which just only one paper reported the existence of aristothiolide, the sulfur-containing phenanthrene derivative,
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in Aristolochia argentina of the genus Aristolochia before [36].
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Compounds 6, 8, 11, 12, 15 and 16 had been tested for the cytotoxicity against HK-2 cell lines in previous study, and compounds 6, 8, 12, 15 and 16 had been proved that they showed varying degree of cytotoxicity
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against HK-2 cells [32, 38]. However, other phenanthrene derivatives obtained from this drug have not been tested whether they have nephrotoxicity. Thus, the cytotoxicity against human renal proximal tubular epithelial
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cell lines (HK-2) of compounds 1, 2, 9, 10, 13 and 14 were investigated by MTT and LDH assays (Table 2). The results of MTT assay showed that compounds 1 and 10 have stronger cytotoxic effect than the positive control (aristololactam I, IC50 76.71 μmol/L). The IC50 values of compounds 1 and 10 were 18.18 and 20.44 μmol/L, respectively. Compound 9 showed moderate cytotoxicity with IC50 values of 95.60 μmol/L, whereas compounds 2, 13 and 14 didn’t exhibit cytotoxicity. In LDH leakage assay, compound 10 also exhibited significant cytotoxicity with IC50 value of 35.06 μmol/L, which was close to that of 28.10 μmol/L of aristololactam I. The IC50 value of 84.36 μmol/L indicated that compound 1 had weak cytotoxicity. However, the other four compounds, namely 2, 9, 13 and 14, showed cytotoxic
effect in neither MTT assay nor LDH assay. In - 13 -
ACCEPTED MANUSCRIPT summary, based on the results of MTT assay in combination with LDH leakage assay, compounds 1 and 10 exhibited strong potential cytotoxicity against human renal proximal tubular epithelial cell lines. Considering the other nephrotoxic phenanthrene derivatives (6, 8, 12, 15 and 16) of the roots and rhizomes of A. heterotropoides var. mandshuricum previously tested, the results implied the potency of renal toxicity of this herb used as a medicine. Compounds 1 , 9 and 10 are the derivatives of 7-methoxy-aristololactam IV (8), aristololactam I (6) and
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aristololactam IV (7), respectively, in which the hydrogen atom are replaced by an ethoxyl at C-9. Compounds 6 and 8 had been proved to be strongly cytotoxic [32, 38], while no literature report the cytotoxic effect of compound 7. Compound 9 didn’t show the strong cytotoxic effect, but compound 1 and 10 remained high
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cytotoxicity. So, it was uncertain whether the ethoxyl at C-9 position exhibited a significant influence on cytotoxic activities, and the explanation of this result needs further investigation. Compounds 2, 13 and 14 were
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all glycosylated at the nitrogen atom of the lactam unit, and they all showed no cytotoxicity. Consequently, it could be deduced from their structures that the glucose unit linked to nitrogen atom might decrease the renal
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cytotoxicity of some aristololactams. This research has proved that nephrotoxic phenanthrene derivatives exist in
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the roots and rhizomes of A. heterotropoides var. mandshuricum. The renal cytotoxicity of this crude drug and other plants of the same genus, which are commonly used in decoction or other types of preparations, should be
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further evaluated in order to ensure the safety of clinic application.
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Acknowledgment.
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This research was financially supported by the National Natural Science Foundation of China (grant no. 81274073 and 81173494), the 985 Project of Peking University and the National Eleventh Five-year Key Technologies R&D Program of China (No. 2006BAI14B01).
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ACCEPTED MANUSCRIPT [46] Mahmood K, Cheong CK, Park MH, Han NY, Han BH. Aristolactams of Orophea enterocarpa. Phytochemistry 1986;25:965–7. [47] Zhu HP, Lu XL, Sun XH, Xu QZ, Jiao BH. Dihydrochalcones and ohenanthrene derivatives from Fissistigma
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bracteolatum. Journal of Medical Colleges of PLA 2010;25:226–34.
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Fig. 1. Structures of compounds 1－16
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ACCEPTED MANUSCRIPT
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Fig. 2. Key HMBC correlations of compounds 1－5.
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Table 1 NMR spectroscopic data of compounds 1, 3, 4 and 5.
1′
—
118.1 105.9 148.3 147.1 109.0 127.9 119.8 126.8 112.3 157.1 120.1 133.1 124.6 125.8 167.3 103.2 — — 56.9 — — — — — — — — — —
— 7.66, s — — — — 8.30, d (8.0) 7.58, t (8.0) 7.30, d (8.0) — — — — — — 6.46, s — — 3.95, s — — 9.88, s — — — — — — —
—
104.6
4.92, d (7.5)
d
121.7 115.1 146.1 141.2 120.6 127.7 119.8 125.0 107.0 155.0 123.9 98.0 134.6 125.6 168.6
2′ 3′ 4′ 5′
— — — —
— — — —
73.9 e 75.9 f 69.7 76.4
6′
—
—
68.4
1′′ 2′′ 3′′ 4′′
— — — —
— — — —
103.0 d 73.0 e 75.8 f 69.8
(J in Hz)
— 7.85, s — — — — 8.99, brs 7.49, t (8.0) 7.16, d (8.0)
δC
— — — 55.7 — — — — — — — — — — 104.1
4.07－2.86, m, H-2′－6′
73.4 75.7 69.8 77.3 60.8
4.09, d (7.5) 4.07－2.86, m, H-2′′－6′′
— — — —
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a
b
δH (J in Hz) — 7.75, s — — — — 8.01,d (2.0) — 7.06, d (2.0) — — 8.14, s — — — 6.55, d (23.5) — — 4.01, s — — —
— — — — — — —
124.4 105.6 150.4 148.9 120.0 127.7 104.9 157.7 100.0 156.4 118.3 117.2 126.7 132.7 190.1 104.5 — — 56.3 — — — — — — — — — —
4.82, d (8.0)
100.5
5.17, d (7.5)
3.43—3.18, m, H-2′－5′
72.1 87.8 68.3 77.1
3.58, m 3.54, m 3.40, m 3.53, m 3.72 , m 3.42 , m 4.40, d (7.5) 3.12, m 3.20, m 3.07, m
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— 7.57, s — — — — 7.96, s — — — — — — — — 6.43, s 3.87, s 3.94, s 3.83, s 4.03, q (7.0) 1.40, t (7.0) 10.91, s — — — — — — —
δC
5
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in Hz)
b δH
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δC
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δH (J in Hz)
4 a
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b δH (J
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c
1 125.8 2 105.4 3 147.9 4 146.4 4a 108.8 4b 123.4 5 105.3 6 151.5 7 143.3 8 150.3 8a 119.2 9 133.7 10 118.5 c 10a 124.2 C=O 167.4 OCH2O 103.4 OCH3-6 55.7 OCH3-7 60.9 OCH3-8 62.4 OCH2 70.2 CH3 15.1 — NH — OH-2′ OH-4′ — OH-6′ — OH-2′′ — OH-3′′ — OH-4′′ — — OH-6′′ glucose moiety
a
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δC
3 b
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1 a
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position
— — 7.46, s
— — — — — — 3.99, s — — 10.59, brs
3.75, dd (12.0, 3.0), H-6′a 3.53, m, H-6′b — — — —
61.3 104.1 74.0 77.1 70.3
5.51, brs 4.82, d (2.5) 4.61, t (5.5) 5.09, d (3.0) 5.02, d (4.5) 4.99, d (5.5) 4.70, t (5.5)
ACCEPTED MANUSCRIPT 5′′
—
—
76.6
—
6′′
—
—
60.8
—
a
— —
76.3 60.6
3.18, m 3.77, m 3.42, m
Recorded at 125 MHz in DMSO-d6. Recorded at 500 MHz in DMSO-d6. Assignments may be interchanged in the same column.
b c-f
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Table 2
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Cytotoxic activities of compounds 1, 2, 9, 10, 13 and 14 isolated from Asarum heterotropoides var.
The value of IC50 was calculated by the values of inhibitions at ten concentrations of each tested compound, 0.0625, 0.25, 0.5, 1, 2.5,
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a
IC50aof LDH μmol/L 84.36 110.15 152.33 35.06 158.45 151.19 28.10
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1 2 9 10 13 14 aristololactam I b
IC50aof MTT μmol/L 18.18 1881.70 95.60 20.44 122.24 >2000 76.71
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Compounds
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mandshuricum against HK-2 Cells.
b
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5, 10, 20, 40 and 80 μg/ml. aristololactam I was used as positive control, and the concentration of it was same to the tested compounds.
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Graphical abstract
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Yu Jing, Yi-Fan Zhang, Ming-Ying Shang, Jie Yu, Jia-Wei Tang, Guang-Xue Liu, Yao-Li Li, Xiao-Mei Li, Xuan Wang, Shao-Qing Cai PII: DOI: Reference:
S0367-326X(16)30615-3 doi: 10.1016/j.fitote.2017.01.008 FITOTE 3557
To appear in:
Fitoterapia
Received date: Revised date: Accepted date:
21 October 2016 16 January 2017 21 January 2017
Please cite this article as: Yu Jing, Yi-Fan Zhang, Ming-Ying Shang, Jie Yu, Jia-Wei Tang, Guang-Xue Liu, Yao-Li Li, Xiao-Mei Li, Xuan Wang, Shao-Qing Cai , Phenanthrene derivatives from roots and rhizomes of Asarum heterotropoides var. mandshuricum. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Fitote(2017), doi: 10.1016/j.fitote.2017.01.008
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Phenanthrene Derivatives from Roots and Rhizomes of Asarum heterotropoides var. mandshuricum
The State Key Laboratory of Natural and Biomimetic Drugs,
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a
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Xuan Wang a, Shao-Qing Cai a, **
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Yu Jing a, c, Yi-Fan Zhang a, Ming-Ying Shang a, *, Jie Yu a, Jia-Wei Tang b, Guang-Xue Liu a, Yao-Li Lia, Xiao-Mei Li b,
Peking University Health Science Center, No.38, Xueyuan Road, Beijing 100191, China
Renal Division, Department of Medicine, First Hospital, and Institute of Nephrologist, Peking University, The Key
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b
Tonghua Gold-horse Pharmaceutical Group Co., Ltd., Beijing 100025, China
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C
China
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Laboratory of Renal Disease, The Ministry of Health of China, Beijing 100034 ,
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* Corresponding author. Tel./Fax: +86 10 8280 2534.
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** Corresponding author. Tel./Fax: +86 10 8280 1693.
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E-mail address: [email protected] (M,-Y. Shang), [email protected] (S, -Q. Cai).
Abstract Five new phenanthrene derivatives: 9-ethoxy-7-methoxy-aristololactam IV (1), norcepharadione A N-β-D-glucopyranoside (2), aristololactamoside I (3), aristololactamoside II (4) and aristothiolactoside (5) together with eleven known phenanthrene derivatives (6—16) were isolated from the ethanol extract of the roots and rhizomes of Asarum heterotropoides var. mandshuricum. The aristololactams with substitution of ethoxy at C-9 position (1, 9, and 10) and the sulfur-containing phenanthrene derivative (5) were reported in the genus Asarum for the first time. Furthermore，six
phenanthrene glucoside derivatives (2—5, 13 and 14) -1-
ACCEPTED MANUSCRIPT were also found in this genus for the first time and compounds 7 and 9—15 were isolated from the genus Asarum for the first time. Six of them (1, 2, 9, 10, 13 and 14) were submitted to cytotoxicity test against human renal proximal tubular epithelial cell lines (HK-2) using MTT and LDH assays. Compounds 1 and 10 showed significant cytotoxic activity against HK-2 cell lines with IC50 values of 18.18 and 20.44 μmol/L in MTT assay and 84.36 and 35.06 μmol/L in LDH assay, respectively. Compound 9 showed moderate cytotoxicity in MTT assay with IC50 values of 95.60 μmol/L, but no cytotoxicity in LDH assay. Compounds 2, 13 and 14 showed cytotoxic effect in
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neither MTT assay nor LDH assay. Considering the other nephrotoxic phenanthrene derivatives (6, 8, 12, 15 and 16) previously tested, the results implied the potency of renal toxicity of this herb used as a medicine.
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Keywords: Asarum heterotropoides var. mandshuricum; Phenanthrene derivatives; Aristolochic acid;
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Aristololactam; Nephrotoxicity;
Chemical compounds studied in this article
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Aristololactam I (PubChem CID: 96710); Aristololactam IV (PubChem CID: 5319404); 9-ethoxy-aristololactam
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I (PubChem CID: 195368); Aristolochic Acid IVa (PubChem CID: 161218); Cepharadione A (PubChem CID: 94577 ); Aristolochic acid I (PubChem CID: 2236)
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1. Introduction
Three classes of phenanthrene derivatives, namely aristolochic acids, aristolactams and 4, 5-dioxoaporphines,
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are a small group of compounds primarily found in Aristolochiaceae [1]. Up to now, aristolochic acids mainly
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occur in the genus Aristolochia and Asarum [2], while aristololactams and 4, 5-dioxoaprophine derivatives are widely found in Annonaceae [1, 2], Saururaceae [3], Menispermaceae [4] andPiperaceae [5]. Aristolochic acids and aristololactams were shown to possess immunostimulatory and anti-inflammatory as well as nephrotoxic, carcinogenic, and mutagenic activities [1]. But they are paid most close attention in recent years among known phenanthrene derivatives for the reason that some of them are associated with a high risk of nephrotoxicity and upper urinary tract carcinoma (UUC), which lead to cause Aristolochic Acid Nephrophathy (AAN) [6–8]. As previously reported，aristolochic acid I is responsible for the destructive fibrotic process in the kidney, which leads to permanent kidney damage in a short period because of aristolochic acid derived-DNA adducts [9].
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ACCEPTED MANUSCRIPT Moreover, aristololactam I was also proved to have cytotoxicity against human proximal tubular cells (HK-2) in recent researches of our group [10, 11]. The genus Asarum contains about 70 species growing in both temperate and tropical regions, and a number of Asarum species are used in herbal medicines throughout the world. Up to date, plants belonging to the genus Asarum were reported to contain a few of phenanthrene derivatives, including aristololactam I [12–17], 5-methoxyaristololactam I [16], 9-hydroxyaristololactam I [15], aristololactam Ia [13], aristololactam II [13],
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aristololactam AII [13, 18], aristololactam III [18], 7-methoxy-aristololactam IV [12–15, 17, 19, 20],
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aristololactam VII [21], aristolochic acid I [12, 13, 15, 16, 22], 7-hydroxyaristolochic acid I [15, 16], aristolochic acid Ia [15], aristolochic acid IV [15], aritolochic acid IVa [14, 17], 4-demethoxyaristolochic acid BII [15], aristololide [13]，aristolic acid I [13], aristolic acid II [15]. The amount of phenanthrene derivatives of the genus
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Asarum is significantly less than that found in the genus Aristolochia [1, 2].
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The root and rhizome of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag is one of the most important crude drug of the genus Asarum in traditional Chinese medicine, which is described as Asari
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Radix et Rhizoma (Xixin) in the Chinese Pharmacopoeia (2010 edition) used as an anodyne, antitussive and
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anti-allergic remedy. It is widely used to treat various diseases such as aphthous stomatitis, headache, toothache and inflammatory in China, Korea and Japan [23]. Although a number of constituents have been isolated, such as
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lignans [24, 25], amides [26], terpenes [27] and flavones [28], only three phenanthrene derivatives including aristololactam I，7-methoxy-aristololactam I and aristolochic acid IVa were isolated from this crude drug [14, 17].
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And a few papers reported that trace amounts (42.2 μg/g, 16.8μg/g, 26.47μg/g) of aristolochic acid I could be
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detected in this crude drug [29–31].
In this paper we focused our interest on phenanthrene derivatives of this crude drug and nephrotoxicity of this kind of compounds. As a result, sixteen phenanthrene derivatives (1—16) containing eleven aristololactams, two aristolochic acids, two 4, 5-dioxoaporphines and one sulfur-containing phenanthrene derivative were isolated and identified from this crude drug. In order to evaluate nephrotoxicity of them, six constituents (1, 2, 9, 10, 13 and 14) were submitted to cytotoxicity test against HK-2 cells using MTT and LDH assays. With the aim of replenishing previous researches on phenanthrene derivatives from the genus Asarum, we reported the isolation, as well as the structure elucidation and nephrotoxicity evaluation of phenanthrene derivatives from Asarum
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ACCEPTED MANUSCRIPT heterotropoides var. mandshuricum. The results can provide us an important clue whether this crude drug can be applied safely in clinic or not. 2. Materials and methods 2.1. General experimental procedures Melting points were obtained on an XT-4A micromelting point apparatus without correction. Optical rotations were determined on a Perkin-Elmer 243B digital polarimeter. UV spectra were carried out on a Cary 300 UV-Vis
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spectrophotometer. A Nicolet NEXUS-470 FTIR spectrophotometer was used for scanning IR spectroscopy.
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NMR spectra were recorded on Varian INOVA-500. The chemical shifts are expressed as δ (ppm) values using solvent as an internal standard, and coupling constant, J, are in Hz. Mass spectra were detected with Bruker APEX IV FT and ABI Q-STAR mass spectrometers. GC was carried out on a Shimadzu GC-2010 series system
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fitted with a FID detector and performed with a DB-1701 column (30 m × 0.25 mm i.d., 0.25 μm film thickness).
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Semi preparative HPLC was conducted on an Alltima C18 column (10 mm i.d. × 250 mm, 10 μm) equipped with an Alltech 426 HPLC pump and an Alltech UVIS 2000 detector. Column chromatography were performed with
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Silica gel (200—300 mesh, Qingdao Marine Chemical Co., Ltd.), Sephadex LH-20 gel (Pharmacia Co., Ltd.). TLC analysis was performed on silica gel (400 mesh, Qingdao Marine Chemical Co., Ltd.) and precoated
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polyamide plates (0.2 mm, Zhejiang Siqing biochem Co., Ltd.) plates. D-glucose was obtained from Beijing
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Chemical Reagent Company. All other chemical solvents used for isolation were of analytical grade (Beijing Beihua Fine Chemicals Co., Ltd.). Fractions were monitored by TLC and spots were detected by UV
2.2. Plant material
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illumination.
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The roots and rhizomes of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. were purchased from Benxi city, Liaoning province, China, in May 2005, and authenticated by Professor Shao-Qing Cai of Peking University Health Science Center. Voucher specimen (No. 5066) was deposited in the Herbarium of Pharmacognosy, School of Pharmaceutical Sciences, Peking University, Beijing, China. 2.3. Extraction and isolation The air-dried and powdered roots and rhizomes of A. heterotropoides var. mandshuricum (36 kg) were extracted three times (2 h, 1.5 h, 1.5 h for each) under reflux with 95% ethanol and then three times (2 h, 1.5 h, 1.5 h for each) with 50% ethanol successively. The
combined extracts were concentrated under reduced -4-
ACCEPTED MANUSCRIPT pressure to give a dark brown extract (9.2 kg), then 7.8 kg of it was suspended in H2O (16.5 L) and partitioned sequentially with petroleum ether (60—90 ℃) (4 × 5 L), CHCl3 (4 × 5 L), EtOAc (4 × 5 L), and n-BuOH (saturated with water) (4 × 5 L), respectively. The CHCl3 layer (231 g) was fractionated on silica gel column chromatography eluting with a gradient of petroleum ether—EtOAc (10:1 to 0:1) to obtain sixteen fractions C1—C16. Fraction C10 and C11 were left to stand overnight and yellow powders as precipitate were collected respectively, then each yellow powder was purified by Sephadex LH-20 eluting with CHCl3—MeOH (6:4) to
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afford compounds 6 (amount 57.8 mg, yield 2.19 μg/g) and 7 (amount 28.5 mg, yield 1.08 μg/g). The residue of
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fraction C11 was subjected to silica gel column chromatography eluting with a gradient of CHCl3—MeOH (1:30 to 1:35) to yield four subfractions C11-1—C11-4. C11-1 yielded a yellow powder, collected by filtering, which showed two high yellow fluorescence spots on TLC under UV illumination. Then the yellow powder was
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separated by semi-preparative HPLC eluting with a gradient of MeOH—H2O to yield compounds 8 (amount 10.2
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mg, yield 0.39 μg/g) and 10 (amount 8.7 mg, yield 0.33 μg/g). The residue of C11-1 was then subjected to chromatography on silica gel eluting with petrol—Me2CO to yield seven tertiary fractions (C11-1-1—C11-1-7).
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C11-1-1 was further separated and purified by Sephadex LH-20 eluting with CHCl3—MeOH (1:1) and recrystallized from MeOH to afford compounds 1 (amount 6.1 mg, yield 0.23 μg/g) and 9 (amount 5.1 mg, yield
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0.19 μg/g); Fraction C12 was chromatographed on silica gel with a gradient of CHCl3－MeOH to give four
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subfractions C12-1－C12-4. C12-3 was further divided into five subfractions C12-3-1－C12-3-5 with repeated
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silica gel column chromatography eluting with CHCl3－MeOH (80:1 to 15:1). C12-3-3 was subsequently further separated on silica gel column with CHCl3—EtOAc and purified by semi-preparative HPLC using a gradient of
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MeOH—H2O as eluent to afford compound 12 (amount 4.2 mg, yield 0.16 μg/g). Fraction C13 was chromatographed on a column packed with silica gel and eluted with CHCl3—MeOH, and subsequently purified by recrystallization from MeOH to afford compound 15 (amount 3.6 mg, yield 0.14 μg/g). The EtOAc layer (50 g) was fractionated on silica gel CC eluting with a gradient of CHCl3－MeOH (10:1 to 0:1) to yield ten fractions E1—E10. Fraction E2 gave yellow powder, which washed with MeOH to afford compound 11 (amount 120 mg, yield 4.37 μg/g). Fraction E3 gave yellow powder which was further separated and purified by semi-preparative HPLC with MeOH—H2O as eluent to yield compound 16 (amount 2.3 mg, yield 0.08 μg/g). Fraction E6 yielded yellowish green flaky crystal, which was further washed with MeOH to afford compound 13 (amount 160 mg, -5-
ACCEPTED MANUSCRIPT yield 5.82 μg/g); Fraction E8 was separated on silica gel column chromatography eluting with CHCl3－MeOH－ H2O (8:1:0.1, 5:1:0.1) and then purified by semi-preparative HPLC with MeOH－H2O as eluent to yield compounds 14 (amount 7.0 mg, yield 0.25 μg/g) and 4 (amount 2.3 mg, yield 0.08 μg/g). The n-BuOH layer (400 g) was fractionated on silica gel column chromatography eluting with a gradient of CHCl3－MeOH (7:1 to 0:1) to yield fourteen fractions B1—B14. Fraction B6 was further separated on silica gel CC eluting with CHCl3－
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MeOH－H2O (5:1:0.1 to 2:1:0.1) to give nine subfractions B6-1－B6-9. B6-4 was subjected to repeated silica gel
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column chromatography eluting with CHCl3－MeOH－H2O (6:1:0.1), followed by semi-preparative HPLC with a gradient of MeOH－H2O as eluent to afford compounds 2 (amount 5.6 mg, yield 0.21 μg/g) , 3 (amount 2.1 mg, yield 0.08 μg/g) and 5 (amount 3.5mg, yield 0.13 μg/g).
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9-ethoxy-7-methoxy-aristololactam IV (1): yellow needles (MeOH); mp 248—249 ℃; UV (MeOH) λmax 238, 288, 299, 338, 403 nm; IR (KBr) νmax 3432—3165, 1690, 1604, 1506, 1473, 1406, 1357, 1305, 1271, 1163, 1130,
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1049, 949 cm-1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) spectroscopic data are
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shown in Table 1; EIMS m/z 397 [M]+ (100), 368 (41), 353 (31), 338 (22), 325 (19), 310 (24), 282 (13), 268 (17), 239 (13), 184 (18); HRESIMS m/z 398.1230 [M + H]+ (calcd. for C21H20NO7, 398.1234).
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Norcepharadione A N-β-D-glucopyranoside (2): red amorphous powder; [α]20D —15.7 (c 1.40, DMSO); UV
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(MeOH) λmax 217, 235, 279, 302, 314, 436 nm; IR (KBr) νmax 3447, 3421, 1647, 1595, 1416, 1365, 1041 cm-1; 1H NMR (DMSO-d6, 500 MHz) δ 8.97 (1H, m, H-11), 8.42 (1H, s, H-7), 8.02 (1H, s, H-3), 8.00 (1H, m, H-8), 7.74
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(2H, m, H-10, 9), 6.60 (2H, s, -OCH2O-), 6.26 (1H, d, J = 10.0 Hz, H-1′), 4.07—3.36 (6H, m, H-4′, 6′, 2′, 3′, 5′); C NMR (DMSO-d6, 125 MHz) δ 173.6 (C-4, C=O), 156.8 (C-5, C=O), 151.4 (C-1), 147.4 (C-2), 131.0 (C-7a),
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129.2 (C-8), 129.1 (C-6a), 128.2 (C-10), 127.6 (C-9), 125.9 (C-11), 124.6 (C-11a), 122.5 (C-3a), 121.4 (C-11c), 117.5 (C-7), 114.2 (C-11b), 107.7 (C-3), 103.7 (-OCH2O-), 85.0 (C-1′), 80.5 (C-5′), 77.5 (C-3′), 69.3 (C-2′), 68.8 (C-4′), 60.6 (C-6′); ESIMS m/z 454 [M + H]+, 476 [M + Na]+; HRESIMS m/z 476.0967 [M + Na]+ (calcd. for C23H19NO9Na, 476.0952). Aristololactamoside I (3): yellow amorphous powder; [α]20D —66.6 (c 0.30, DMSO); UV (MeOH) λmax 210, 240, 263, 285, 329, 349, 404 nm; IR (KBr) νmax 3415, 1673, 1627, 1466, 1406, 1304, 1046, 612 cm-1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) spectroscopic data are shown in Table 1; ESIMS m/z 634 [M + H]+, 656 [M + Na]+; HRESIMS m/z
634.1771 [M + H]+ (calcd for C29H32NO15, 634.1766). -6-
ACCEPTED MANUSCRIPT Aristololactamoside II (4): yellow amorphous powder; [α]20D —28.3 (c 0.46, DMSO); UV (MeOH) λmax 212, 256, 286, 326, 383 nm; IR (KBr) νmax 3393, 1663, 1545, 1467, 1416, 1374, 1288, 1258, 1030, 628 cm-1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) spectroscopic data are shown in Table 1; ESIMS m/z 444 [M + H]+, 466 [M + Na]+, 909 [2M + Na]+; HRESIMS m/z 444.1281 [M + H]+ (calcd for C22H22NO9, 444.1289). Aristothiolactoside (5): yellow amorphous powder; [α]20D —30.0 (c 0.70, DMSO); UV (MeOH) λmax 251, 272,
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294, 310, 341, 361, 416; nm; IR (KBr) νmax 3420, 2920, 1663, 1613, 1461, 1424, 1357, 1275, 1077, 1047, 638
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cm-1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) spectroscopic data are shown in Table 1; ESIMS m/z 651 [M + H]+, 673 [M + Na]+; HRESIMS m/z 651.1399 [M + H]+ (calcd for C29H31SO15,
2.4 Acid Hydrolysis of Compound 2－5, and GC Analysis
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651.1378).
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Compound 2－5 (each about 1 mg) in 2 N HCl (3 ml) were refluxed at 85 ℃ for 3 h, respectively. After cooling, the reaction mixture was neutralized with NaHCO3 and successively extracted with EtOAc (4 × 3 ml). The
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aqueous layer was then evaporated to dryness and the residue was dissolved in anhydrous pyridine (200 μl), and
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L-cysteine methyl ester hydrochloride (0.06 mol/L, 200 μl) was added. The mixture was stirred at 60 ℃ for 1 h.
Then 150 μl trimethylsilylation reagent hexamethyldisilazane—trimethyl chlorosilane (HMDS—TMCS, 3:1) was
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added, and the mixture was stirred at 60 ℃ for an additional 30 min. After centrifugation, the supernatant was concentrated under a stream of N2. The residue was portioned between n-hexane and H2O (0.2 ml each), and the
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n-hexane layer (2 μl) was analyzed by GC for sugar identification (detector, FID; injection temperature, 260 ℃;
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detector temperature, 280 ℃; temperature gradient system for the oven, 160 ℃ for 1 min and then raised to 230 ℃ at rate of 5 ℃/min; carrier gas, N2; flow rate, 1 mL/min). D-glucose were identified for compounds 2－5 by comparion with retention time of authentic D-glucose (tR = 20.76 min) after treatment in the same manner. 2.5 Cytotoxicity Assay in HK-2 Cell Lines. Cytotoxicity was assessed by two methods as described previously [32], including LDH leakage into culture medium, which indicates the integrity of cellular membrane, and MTT conversion into insoluble formazan stain, which shows functional mitochondrial dehydrogenase. All experiments were performed with HK-2 cells, which were obtained from American Tissue Culture Collection (Rockville, Md., USA). All tested compounds were -7-
ACCEPTED MANUSCRIPT dissolved in dimethylsulfoxide (DMSO) (Sigma), and then diluted in Dulbeccos′s modified Eagle′s medium (DEME) (Gibco Invitrogen Corp.) to the indicated concentration of experiments. Results and Discussion The EtOH extract of the roots and rhizomes of Asarum heterotropoides var. mandshuricum was suspended in H2O and partitioned with petroleum ether, CHCl3, EtOAc and n-BuOH, successively. The CHCl3, EtOAc and n-BuOH extracts were separated and purified by various column chromatographic methods to obtain sixteen
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phenanthrene derivatives, including five new compounds (1—5) and eleven known compounds (6—16) (Fig. 1).
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Their structures were elucidated based on the spectroscopic methods. The eleven known compounds were identified as aristololactam I (6) [33], aristololactam IV (7) [34], 7-methoxy-aristololactam IV (8) [33], 9-ethoxy-aristololactam I (9) [35], 9-ethoxy-aristololactam IV (10) [36], aristolochic acid IVa (11) [37],
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aristololactam IVa (12) [38], aristololactam II N-β-D-glucopyranoside (13) [39], aristololactam Ia
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N-β-D-glucopyranoside (14) [40], cepharadione A (15) [41] and aristolochic acid I (16) [33] by comparison of the spectroscopic data with those reported in the literatures. Moreover, compounds 7 and 9—15 were isolated from
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the genus Asarum for the first time.
Compound 1 was obtained as a yellow needle. The molecular formula was deduced as C21H19NO7 by its
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HRESIMS (m/z 398.1230 [M + H]+, calcd. 398.1234). Its UV spectrum in methanol showed characteristics of a
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phenanthrene derivative at 238, 288, 299, 338 and 403 nm [42]. The IR spectrum showed absorptions for imino group (3432—3165 cm-1) and lactamic carbonyl group (1690 cm-1). The 1H NMR spectrum of 1 showed the
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presence of three methoxyls δ 3.94 (3H, s), 3.87 (3H, s) and 3.83 (3H, s). The two singlet signals of aromatic protons at δ 7.96 (1H, s) and 7.57 (1H, s) assignable to H-5 and H-2, respectively, which was confirmed by the
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correlation between H-5 and C-7, C-4a, and C-8a, H-2 and C-10a, C-4, and carbonyl carbon in HMBC experiment. The methylenedioxy group was observed as a singlet at δ 6.43 (2H, s). The other singlet at δ 10.91 (1H, s) was assigned to NH. The 1H NMR spectrum showed the presence of an oxygenated methylene signal at δ 4.03 (2H, q, J = 7.0 Hz) and a methyl signal at δ 1.40 (3H, t, J = 7.0 Hz), which were attributed to an ethoxyl group. This was further supported by the correlations of the protons of above two groups in 1H-1H COSY spectrum. The HMBC experiment showed the correlation between the protons of the oxygenated methylene group at δ 4.03 and C-9 (Fig. 2), so the ethoxyl group should be located at C-9. The 1H, 13C NMR and DEPT
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ACCEPTED MANUSCRIPT spectra showed the expected one methyl, two methines, thirteen quaternary carbons, three methoxyl groups, one methylenedioxy group, and one oxygenated methylene group. According to 1H and 13C NMR spectra, the only difference between compound 1 and 9-ethoxy-aristololactam IV (10) [36] was the presence of one methoxyl at δ 3.94 (3H, s) attached to C-7. The full assignments of 1H and 13C NMR data of compound 1 was achieved by comparing them with those of 9-ethoxy-aristololactam I (9) [35] and 9-ethoxy-aristololactam IV (10), which
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compound 1 was established as 9-ethoxy-7-methoxy-aristololactam IV.
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further assigned by using an HMBC experiment (Fig. 2). On the basis of the above spectroscopic analysis,
Compound 2 was obtained as a red amorphous powder. The molecular formula of compound 2 was established to be C23H19NO9 by HRESIMS (m/z 476.0967 [M + Na]+, calcd. 476.0952). The UV spectrum showed absorptions
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of a 4, 5-dioxoaporphine derivative at 217, 235, 279, 302, 314 and 436 nm [43]. The presence of hydroxyl and carbonyl groups were supported by the IR spectrum peaks at 3447—3421 and 1647 cm-1. The 1H NMR spectrum
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didn’t show the presence of methoxyl and NH groups. In the 1H NMR spectrum, one set of four mutually coupled proton signals at δ 8.97 (1H, m), 8.00 (1H, m) and 7.74 (2H, m) corresponded to H-11, H-8, H-10 and H-9,
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respectively. The singlet signals at δ 8.02 (1H, s) and 8.42 (1H, s) were assigned to H-3 and H-7, respectively,
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which were inferred by the HMBC correlations between H-3 and C-1, C-11c, and H-7 and C-11a, C-11c (Fig. 2). The singlet signal at δ 6.60 (2H, s) was assigned to a methylenedioxy, because the protons showed the long-range
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correlations to C-1 and C-2 in HMBC spectrum. In the 1H NMR spectrum of compound 2 a doublet signal at δ
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6.26(1H, d, J = 10.0 Hz) was assigned as an anomeric proton of glucose moiety. The 1H and 13C NMR data as well as acid hydrolysis and GC comparison with an authentic sample indicated the presence of a moiety. The β-configuration of the glycoside linkage was inferred by its large coupling
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D-glucopyranosyl
constant J = 10.0 Hz of the anomeric proton at δ 6.26 (1H, d). Comparing with N-methoxy norcepharadione A [44], the absence of a methoxyl group in 2 suggested that the glucosyl moiety likely attached to the nitrogen of the aglycone. To confirm the connection between the glucosyl and the phenanthrene unit of compound 2, a HMBC experiment was conducted. The results showed the correlations between the anomeric proton H-1′ and C-6a, C-5 (Fig. 2), which further supported the above conclusion. Therefore, the structure of compound 2 was established to be norcepharadione A N-β-D-glucopyranoside. Compound 3 was obtained as a yellow amorphous
powder. The molecular formula was suggested to be -9-
ACCEPTED MANUSCRIPT C29H31NO15 by HRESIMS (m/z 634.1771 [M + H]+, calcd. 634.1766). The UV spectrum exhibited the characteristic at 240, 263, 285, 329, 349 and 404 nm of a phenanthrene chromophore [42]. The IR bands at 3415 and 1673 cm-1 revealed the presence of hydroxyl and lactamic carbonyl group. The signal for H-2 was observed at δ 7.66, which confirmed by the correlations between H-2 and C-10a, C-4, and carbonyl group in HMBC spectrum (Fig. 2). The aromatic region of the 1H NMR spectrum contained an ABC pattern signals at δ 8.30(1H, d, J = 8.0 Hz), 7.58(1H, t, J = 8.0 Hz) and 7.30(1H, d, J = 8.0 Hz), which was ascribed to H-5, H-6 and H-7, respectively.
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One methoxyl group proposed to be attached to C-8 was further confirmed by HMBC correlations between H-6,
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methoxyl protons (δ 3.95) and C-8. The two singlets at δ 9.88 (1H, s) and 6.46 (2H, s) were attributed to NH and methylenedioxy, respectively. The 1H and 13C NMR data suggested compound 3 was a monomethoxyl aristololactam with 3, 4-methylenedioxy substituent. The 13C NMR singals at δ 104.6, 103.0, 76.6, 76.4, 75.9,
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75.8, 73.9, 73.0, 69.8, 69.7, 68.4 and 60.8 indicated the presence of two glucopyranose moieties [45]. The typical coupling constants of two anomeric protons at δ 4.92 (1H, d, J = 7.5 Hz) and 4.09 (1H, d, J = 7.5 Hz) suggested
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the β-conformations for two glucoses. The acid hydrolysis and GC comparison with an authentic sample
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indicated the presence of a D-glucose. According to above analysis, C-9 is a suitable position available for the glycosidic linkage, which was further supported by the correlation between the anomeric proton H-1′ and C-9 in
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HMBC spectrum (Fig. 2). Furthermore, in 13C NMR spectrum the C-6′ signal in one glucosyl downfield shifted to
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δ 68.4, which indicated that the second anomeric carbon C-1′′ should be linked to C-6′. Moreover, the correlation between H-1′′ and C-6′ in HMBC spectrum also proved the above conclusion (Fig. 2). The combined evidence
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supported the structure of compound 3 for aristololactam I
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9-O-β-D-glucopyrannosyl(1→6)-O-β-D-glucopyranoside, and it was named as aristololactamoside I. Compound 4 was obtained as a yellow amorphous powder. Its molecular formula was established as C22H21NO9 by HRESIMS (m/z 444.1281 [M + H]+, calcd. 444.1289). The UV spectrum of compound 4 showed absorptions at 212, 256, 286, 326 and 383 nm, which corresponded to the characteristic of a phenanthrene chromophore [42]. The IR spectrum indicated the presence of hydroxyl or imino group (3493 cm-1) and lactamic carbonyl group (1663 cm-1). The 1H NMR spectrum revealed the presence of an imino proton at δ 10.59 (1H, s). Through analysis of 1H NMR and 1H-1H COSY spectra, an ABC system aromatic protons signals were observed at δ 8.99 (1H, brs), 7.49 (1H, t, J = 8.0 Hz) and 7.16 (1H, d, J = 8.0Hz), which were assigned to H-5, H-6 and H-7, - 10 -
ACCEPTED MANUSCRIPT respectively. And the correlations between H-7 and C-5, C-8a, H-6 and C-4b in HMBC spectrum further confirmed above conclusions (Fig. 2). The other two singlets at δ 7.85 (1H, s) and δ 7.46 (1H, s) were attributed to H-2 and H-9, respectively, which were supported by the cross peaks between H-2 and C-10a, H-9 and C-4b, C-10a in HMBC spectrum (Fig. 2). The 1H NMR spectrum also showed methoxyl protons signal at δ 3.99 (3H, s). And the correlations between H-9, the methoxyl protons (δ 3.99) and C-8 in HMBC spectrum inferred that the methoxyl attached to C-8. In addition, the 1H and 13C NMR spectra of compound 4 exhibited the existence of
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β-glucopyranose moiety, which supported by coupling constants of the anomeric proton at δ 4.82(1H, d, J = 8.0
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Hz). And acid hydrolysis confirmed the absolute configuration of the sugar moieties as D-glucose as determined by GC analysis. Comparing the 1H and 13C NMR data of compound 4 with those of enterocarpam II [46], we concluded that the glucosyl moiety most likely attached to C-3 (δ 146.1) or C-4 (δ 141.2). In the NOESY
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experiment, there was no NOE correlation between H-2 and the anomeric proton (H-1′). On the other hand, the
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signal for C-4 of compound 4 at δ 141.25 was much more high field than the corresponding carbon signal at δ 145.75 of 10-amino-3, 4-dihydroxyl phenanthrene-1-carboxylic acid lactam [47] due to glycosidation effect of
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phenolic hydroxyl at C-4. Based on the above evidence, we confirmed that the glycosidic linkage of compound 4 was determined to be at C-4. But the HMBC correlation between the anomeric proton and C-4 (δ 141.2) was not
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observered due to the small quality of compound 4. In combination with 1H, 13C NMR and HRESIMS data, it
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could be concluded that a hydroxyl group existed in the structure and attached to C-3. On the basis of the above spectroscopic data, the structure of compound 4 was determined to be enterocarpam II 4-O-β-D-glucopyranoside
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and named as aristololactamoside II.
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Compound 5 was obtained as yellow amorphous powder. The HRESIMS, exhibiting a molecular ion peak at m/z 651.1399 [M + H]+, indicated that the molecular formula was C29H30SO15 (calcd. 651.1378). The UV spectrum exhibited absorption at 251, 272, 294, and 341 nm, which correspongding to a phenanthrene chromophore. The IR spectrum showed absorptions for hydroxyl
(3420 cm-1 )and carbonyl (1663 cm-1) groups,
respectively. Its NMR spectra exhibited the presence of one methoxyl group [δH 4.01 (3H, s) and δC 56.3], one methylenedioxy group [δH 6.55 (2H, d, J = 23.5 Hz) and δH 104.5], two aromatic protons [δH 7.75 (1H, s) and δC 105.6; δH 8.14 (1H, s) and δC 117.2], two meta-coupled aromatic protons [δH 8.01 (1H, d, J = 2.0 Hz) and δC 104.9; δH 7.06 (1H, d, J = 2.0 Hz) and δC 100.0]. Moreover, the 1H NMR spectrum also showed the presence of - 11 -
ACCEPTED MANUSCRIPT two anomeric protons of glucose moiety at δ 5.17 (1H, d, J = 7.5 Hz, H-1′) and δ 4.40 (1H, d, J = 7.5 Hz, H-1′′), together with two sets of protons signal of glucose moiety including seven hydroxyl groups [δ 5.51 (1H, brs), δ 5.09 (1H, d, J = 3.0 Hz), δ 5.02 (1H, d, J = 4.5 Hz), δ 4.99 (1H, d, J = 5.5 Hz), δ 4.82 (1H, d, J = 2.5 Hz), δ 4.70 (1H, t, J = 5.5 Hz) and δ 4.61 (1H, t, J = 5.5 Hz)] and twelve glucosyl protons [δ 3.71—3.79 (2H, m), δ 3.52—3.58 (4H, m), δ 3.39—3.44 (2H, m), δ 3.18—3.24 (2H, m) and δ 3.06—3.12 (2H, m)]. The 13C-NMR and HMQC spectra showed signals for fourteen aromatic carbons, one carbonyl group (δC 190.1), one methylenedioxy
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group (δC 104.5), one methoxyl group (δC 56.3) and twelve glucosyl carbons (δC 104.5, 100.5, 87.8, 77.1,
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77.1,76.3, 74.0, 72.1 70.3, 68.3 61.3 and 60.6). The HRESIMS indicated compound 5 was a sulfur-containing compound. So the carbonyl group (δC 190.1) was lower field than the lactam carbonyl group of aristololactam derivatives due to the deshielding effect of sulfur atom. The 1H and 13C NMR spectroscopic data of compound 5
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were very similar to that reported in the literature for aristothiolide [36], except for the absence of a methoxyl group at C-6. The NMR spectra showed the presence of two glucopyranosyl moieties in compound 5. Acid
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hydrolysis of compound 5 liberated D-glucose, which was determined by GC analysis. And the β-configurations
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of two glucose units were determined from the coupling constant of the two anomeric protons. The location of the inner glucose moiety at C-6 in compound 5 was corroborated by HMBC experiment (Fig. 2) that showed
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correlation between the anomeric proton (δ 5.17, H-1′) and C-6 (δ 157.7). In addition, the connectivities of two
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β-D-glucopyranosyl moieties in compound 5 were clarified by HMBC experiment, in which long-range correlation were observed between anomeric proton (δH 4.40, H-1′′) of the terminal glucose moiety and C-3′ (δ
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87.8) of the inner glucose moiety (Fig. 2). Additionally, the HMBC correlations (Fig. 2) of the aromatic protons H-2 (δH 7.75) with C-4 (δC 148.9), C-10a (δC 132.7) and carbonyl carbon (δC 190.1), H-9 (δH 8.14) with C-3 (δC
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150.4), C-4b (δC 127.7) and C-10a (δC 132.7), H-5 (δH 8.01) with C-4a (δC 120.0), C-8a (δC 118.3) and C-7 (δC 100.0), H-7 (δH 7.06) with C-5(δC 104.9) and C-8a (δC 118.3) as well as the methylenedioxy protons (δH 6.55) with C-3 (δC 150.4) and C-4 (δC 148.9) were observed. On the basis of the above results and spectral data, the structure of compound 5 was recognized as 6-demethy aristothiolide 6-O-β-D-glucopyrannosyl(1→3)-O-β-D-glucopyranoside and named as aristothiolactoside. Compounds 1 as well as the known 9 and 10 all have an ethoxyl group at C-9 position in their structures, which reported in the Asarum plants for the first time. This kind of phenanthrene derivative with an ethoxyl group is
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ACCEPTED MANUSCRIPT extremely rare in nature, for only compounds 9 and 10 had been obtained only from Aristolochia mollisimma Hance. [35] and Aristolochia argentina [36], respectively. There were just sixteen phenanthrene glycosidic derivatives found in the genus Aristolochia of Aristolochiaceae so far, including fourteen monoglycosides and two single-chain diglucosides [1, 2]. However, until now no phenanthrene glycosidic derivative was obtained from the genus Asarum. In this paper we first reported six phenanthrene glycosidic derivatives (2—5, 13 and 14) isolated from Asarum heterotropoides var. mandshuricum of the genus Asarum. Moreover, compound 3 and 5
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time that phenanthrene diglycosidic derivatives were isolated from this genus.
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exhibited a single-chain glycoside consisting of two glucoses in their structures, respectively, which was the first
Compound 4 was isolated as an oxygen glycoside with glycosidic linkage and a hydroxyl group at the C-4 and C-3 position, respectively. To our best knowledge, aristololactams having a methoxyl and a hydroxyl group at
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C-3(4) and C-4(3) positions, two methoxyl groups or methylenedioxy group at C-3 and C-4 positions, are
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common in the plants of Aristolochiaceae [1, 2]. But hitherto there have been no report of aristololactam like compound 4 with the glycosidic linkage at C-4 position having been isolated from nature. In addition, compound
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5 was identified as a rare sulfur-containing phenanthrene glycosidic derivative with aristothiolide as aglycone, which just only one paper reported the existence of aristothiolide, the sulfur-containing phenanthrene derivative,
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in Aristolochia argentina of the genus Aristolochia before [36].
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Compounds 6, 8, 11, 12, 15 and 16 had been tested for the cytotoxicity against HK-2 cell lines in previous study, and compounds 6, 8, 12, 15 and 16 had been proved that they showed varying degree of cytotoxicity
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against HK-2 cells [32, 38]. However, other phenanthrene derivatives obtained from this drug have not been tested whether they have nephrotoxicity. Thus, the cytotoxicity against human renal proximal tubular epithelial
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cell lines (HK-2) of compounds 1, 2, 9, 10, 13 and 14 were investigated by MTT and LDH assays (Table 2). The results of MTT assay showed that compounds 1 and 10 have stronger cytotoxic effect than the positive control (aristololactam I, IC50 76.71 μmol/L). The IC50 values of compounds 1 and 10 were 18.18 and 20.44 μmol/L, respectively. Compound 9 showed moderate cytotoxicity with IC50 values of 95.60 μmol/L, whereas compounds 2, 13 and 14 didn’t exhibit cytotoxicity. In LDH leakage assay, compound 10 also exhibited significant cytotoxicity with IC50 value of 35.06 μmol/L, which was close to that of 28.10 μmol/L of aristololactam I. The IC50 value of 84.36 μmol/L indicated that compound 1 had weak cytotoxicity. However, the other four compounds, namely 2, 9, 13 and 14, showed cytotoxic
effect in neither MTT assay nor LDH assay. In - 13 -
ACCEPTED MANUSCRIPT summary, based on the results of MTT assay in combination with LDH leakage assay, compounds 1 and 10 exhibited strong potential cytotoxicity against human renal proximal tubular epithelial cell lines. Considering the other nephrotoxic phenanthrene derivatives (6, 8, 12, 15 and 16) of the roots and rhizomes of A. heterotropoides var. mandshuricum previously tested, the results implied the potency of renal toxicity of this herb used as a medicine. Compounds 1 , 9 and 10 are the derivatives of 7-methoxy-aristololactam IV (8), aristololactam I (6) and
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aristololactam IV (7), respectively, in which the hydrogen atom are replaced by an ethoxyl at C-9. Compounds 6 and 8 had been proved to be strongly cytotoxic [32, 38], while no literature report the cytotoxic effect of compound 7. Compound 9 didn’t show the strong cytotoxic effect, but compound 1 and 10 remained high
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cytotoxicity. So, it was uncertain whether the ethoxyl at C-9 position exhibited a significant influence on cytotoxic activities, and the explanation of this result needs further investigation. Compounds 2, 13 and 14 were
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all glycosylated at the nitrogen atom of the lactam unit, and they all showed no cytotoxicity. Consequently, it could be deduced from their structures that the glucose unit linked to nitrogen atom might decrease the renal
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cytotoxicity of some aristololactams. This research has proved that nephrotoxic phenanthrene derivatives exist in
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the roots and rhizomes of A. heterotropoides var. mandshuricum. The renal cytotoxicity of this crude drug and other plants of the same genus, which are commonly used in decoction or other types of preparations, should be
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further evaluated in order to ensure the safety of clinic application.
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Acknowledgment.
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This research was financially supported by the National Natural Science Foundation of China (grant no. 81274073 and 81173494), the 985 Project of Peking University and the National Eleventh Five-year Key Technologies R&D Program of China (No. 2006BAI14B01).
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ACCEPTED MANUSCRIPT [46] Mahmood K, Cheong CK, Park MH, Han NY, Han BH. Aristolactams of Orophea enterocarpa. Phytochemistry 1986;25:965–7. [47] Zhu HP, Lu XL, Sun XH, Xu QZ, Jiao BH. Dihydrochalcones and ohenanthrene derivatives from Fissistigma
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Fig. 1. Structures of compounds 1－16
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Fig. 2. Key HMBC correlations of compounds 1－5.
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Table 1 NMR spectroscopic data of compounds 1, 3, 4 and 5.
1′
—
118.1 105.9 148.3 147.1 109.0 127.9 119.8 126.8 112.3 157.1 120.1 133.1 124.6 125.8 167.3 103.2 — — 56.9 — — — — — — — — — —
— 7.66, s — — — — 8.30, d (8.0) 7.58, t (8.0) 7.30, d (8.0) — — — — — — 6.46, s — — 3.95, s — — 9.88, s — — — — — — —
—
104.6
4.92, d (7.5)
d
121.7 115.1 146.1 141.2 120.6 127.7 119.8 125.0 107.0 155.0 123.9 98.0 134.6 125.6 168.6
2′ 3′ 4′ 5′
— — — —
— — — —
73.9 e 75.9 f 69.7 76.4
6′
—
—
68.4
1′′ 2′′ 3′′ 4′′
— — — —
— — — —
103.0 d 73.0 e 75.8 f 69.8
(J in Hz)
— 7.85, s — — — — 8.99, brs 7.49, t (8.0) 7.16, d (8.0)
δC
— — — 55.7 — — — — — — — — — — 104.1
4.07－2.86, m, H-2′－6′
73.4 75.7 69.8 77.3 60.8
4.09, d (7.5) 4.07－2.86, m, H-2′′－6′′
— — — —
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a
b
δH (J in Hz) — 7.75, s — — — — 8.01,d (2.0) — 7.06, d (2.0) — — 8.14, s — — — 6.55, d (23.5) — — 4.01, s — — —
— — — — — — —
124.4 105.6 150.4 148.9 120.0 127.7 104.9 157.7 100.0 156.4 118.3 117.2 126.7 132.7 190.1 104.5 — — 56.3 — — — — — — — — — —
4.82, d (8.0)
100.5
5.17, d (7.5)
3.43—3.18, m, H-2′－5′
72.1 87.8 68.3 77.1
3.58, m 3.54, m 3.40, m 3.53, m 3.72 , m 3.42 , m 4.40, d (7.5) 3.12, m 3.20, m 3.07, m
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— 7.57, s — — — — 7.96, s — — — — — — — — 6.43, s 3.87, s 3.94, s 3.83, s 4.03, q (7.0) 1.40, t (7.0) 10.91, s — — — — — — —
δC
5
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in Hz)
b δH
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δC
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δH (J in Hz)
4 a
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b δH (J
PT
c
1 125.8 2 105.4 3 147.9 4 146.4 4a 108.8 4b 123.4 5 105.3 6 151.5 7 143.3 8 150.3 8a 119.2 9 133.7 10 118.5 c 10a 124.2 C=O 167.4 OCH2O 103.4 OCH3-6 55.7 OCH3-7 60.9 OCH3-8 62.4 OCH2 70.2 CH3 15.1 — NH — OH-2′ OH-4′ — OH-6′ — OH-2′′ — OH-3′′ — OH-4′′ — — OH-6′′ glucose moiety
a
CE
δC
3 b
M
1 a
AC
position
— — 7.46, s
— — — — — — 3.99, s — — 10.59, brs
3.75, dd (12.0, 3.0), H-6′a 3.53, m, H-6′b — — — —
61.3 104.1 74.0 77.1 70.3
5.51, brs 4.82, d (2.5) 4.61, t (5.5) 5.09, d (3.0) 5.02, d (4.5) 4.99, d (5.5) 4.70, t (5.5)
ACCEPTED MANUSCRIPT 5′′
—
—
76.6
—
6′′
—
—
60.8
—
a
— —
76.3 60.6
3.18, m 3.77, m 3.42, m
Recorded at 125 MHz in DMSO-d6. Recorded at 500 MHz in DMSO-d6. Assignments may be interchanged in the same column.
b c-f
T
Table 2
CR IP
Cytotoxic activities of compounds 1, 2, 9, 10, 13 and 14 isolated from Asarum heterotropoides var.
The value of IC50 was calculated by the values of inhibitions at ten concentrations of each tested compound, 0.0625, 0.25, 0.5, 1, 2.5,
ED
a
IC50aof LDH μmol/L 84.36 110.15 152.33 35.06 158.45 151.19 28.10
AN
1 2 9 10 13 14 aristololactam I b
IC50aof MTT μmol/L 18.18 1881.70 95.60 20.44 122.24 >2000 76.71
M
Compounds
US
mandshuricum against HK-2 Cells.
b
AC
CE
PT
5, 10, 20, 40 and 80 μg/ml. aristololactam I was used as positive control, and the concentration of it was same to the tested compounds.
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ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
US
CR IP
T
Graphical abstract
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