Two Prenylated and C-Methylated Flavonoids from Tripterygium wilfordii

• Abstract
• Abbreviations
• Materials and Methods
• In vitro NO production inhibition assay 21
• In vitro antitumor cell line proliferation assay
• In vitro antimicrobial assay
• X‐ray crystal structure analysis of 1
• Supporting information
• Acknowledgements
• References

Abstract

Two unusual prenylated and C-methylated flavanones, (±)-5,4′-dihydroxy-2′-methoxy-6′,6′′- dimethypyrano-(2′′,3′′:7,8)-6-methyflavanone (1) and (2S)-5,7,4′-trihydroxy-2′-methoxy-8,5′- di(3-methyl-2-butenyl)-6-methyflavanone (2), and 10 known compounds were isolated from the stems and roots of Tripterygium wilfordii. Their structures were elucidated based on spectroscopic analyses including 1- and 2-D NMR, HR‐ESI‐MS, CD, and x-ray crystallography. The anti-inflammatory, antiproliferative, and antibacterial activities of compounds 1 and 2 were investigated. Compound 2 had an inhibitory effect on lipopolysaccharides (LPS)-triggered RAW cell nitric oxide (NO) production, with an IC₅₀ value of 15.0 ± 0.7 µM. Both compounds showed moderate antiproliferative activity against the tumor cell lines HT-29 and ZR-75-1.

Abbreviations

5-FU: 5-fluorouracil

LPS: lipopolysaccharides

NO: nitric oxide

DMEM: Dulbecco's modified Eagle's medium

Tripterygium wilfordii Hook. F. (Celastraceae) grows mainly in the southern part of China. The debarked stems and roots of T. wilfordii, known as “Lei gong teng,” are famous for their potent anti-inflammatory and immune system-regulating activities. In China, Lei gong teng has long been used clinically against autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis, Crohn's disease, and lupus [1], [2], [3], [4]. Previous phytochemical investigations reported the isolation of many sesquiterpenes, diterpenes, and triterpenes from T. wilfordii with anti-inflammatory, antitumor, antifertility, insecticidal, and antineurodegenerative activities [5]. Our ongoing phytochemical study of T. wilfordii resulted in the isolation of two new prenylated and C-methylated flavanones, (±)-5,4′-dihydroxy-2′-methoxy-6′,6′′-dimethypyrano(2′′,3′′:7,8)-6-methyflavanone (1) and (2S)-5,7,4′-trihydroxy-2′-methoxy-8,5′-di(3-methyl-2-butenyl)-6-methyflavanone (2) ([Fig. 1]), along with 10 known compounds, namely, friedelin [6], betulinic acid [7], β-amyrin [8], oleanolic acid [9], kaempferol [10], β-sitosterol, n-hexadecanoic acid [11], methyl hexadecanoate [11], n-octadecanoic acid [12], and n-tritriacontanoic acid [12]. The present paper reports the isolation and structural elucidation of the new prenylated methylflavones as well as an evaluation of their inhibitive activity on LPS-triggered RAW cell NO production, their antiproliferative activity against the tumor cell lines ZR-75-1 and HT-29, and their bacterial inhibition activity against Escherichia coli strain DH5-alpha.

Compound 1 was obtained as a yellow needle-like crystal and appeared as one bright orange spot on the TLC plate when observed with 10 % H₂SO₄−EtOH spraying reagent followed by gentle heating (m. p. 210–212 °C). [α]_D ²⁰ was determined as 0 (c 0.3, MeOH), implying that 1 might be a racemate. The X-ray crystallography analysis demonstrated that a pair of enantiomers exists in one unit cell (see Fig. 23S in Supporting Information). HR‐EI‐MS at m/z = 381.1335 [M – H]⁻ (calcd. for C₂₂H₂₁O₆: 381.1338) and EI‐MS at m/z = 382 [M]⁺ established the formula of 1 as C₂₂H₂₂O₆ with 12 degrees of unsaturation. The IR spectrum showed absorptions of hydroxy and carbonyl groups at 3387 and 1643 cm⁻¹, respectively. A typical flavanone skeleton was shown, with ¹H‐NMR resonances at δ _H = 5.89 (d, J = 13.2 Hz, H-2), 2.97 (d, J = 13.2 Hz, H-3a), and 3.26 (d, J = 17.2, 13.2 Hz, H-3b) and ¹³C‐NMR resonances at δ _C = 43.0 (C-3), 75.18 (C-2), and 198.0 (C-4) [13]. The ¹H−¹H COSY correlations of H-2 with H₂-3 and the HMBCs of H₂-3/C-2, C-1′, C-4, C-5 and H-2/C-2′, C-6′, C-4, C-9 further confirmed the flavanone skeleton ([Fig. 2]). The presence of a chromene ring system was deduced from the gem-dimethyl singlets at δ _H = 1.30 (3H, s) and 1.33 (3H, s), the two doublets at δ _H = 5.51 (d, J = 10.0 Hz H-5′′) and 6.73 (d, J = 10.0 Hz H-4′′), and related HMBCs [14]. The HMBCs of H-2/C-9 (δ _C = 156.2) and H₂-1′′/C-7 (δ _C = 160.2), C-8 (δ _C = 102.2), and C-9 (δ _C = 156.2) revealed that the dimethylpyran group should be fused to C-7 and C-8 of the A ring ([Fig. 2]). A C-methyl group [δ _H = 2.20 (3H, s), δ _C = 7.5 (q)] at C-6 and a hydroxy group at C-5 were determined by the HMBC cross-peaks of a Me (δ _H = 2.44) with C-7 (δ _C = 160.6), C-6 (δ _C = 104.7), and C-5 (δ _C = 164.1). A methoxy group at C-2′ was identified by the HMBCs of H-2/C-1′, C-2′, C-6′, and H₃-MeO/C-2′. Another hydroxy group at C-4′ was evident from an ABX system in the B ring with signals at δ _H = 6.88 (br s, H-3′), 6.93 (d, J = 8 Hz, H-5′), and 7.63 (d, J = 8 Hz, H-6′). The above deduction was in good agreement with the X-ray crystallography data analysis as the ORTEP‐III view of the (2R) enantiomer shown in [Fig. 3]. Therefore, 1 was identified as (±)-5,4′-dihydroxy-2′-methoxy-6′,6′′-dimethypyrano(2′′,3′′:7,8)-6-methyflavanone. It might be derived from (±)-5,7,4′-trihydroxy-2′-methoxy-8-(3-hydroxy-3-methyl-1-butenyl)-5′-(3-methyl-2-butenyl)-6-methyflavanone biogenetically or by losing a molecule of water to form a pyran ring during extraction and isolation. To the best of our knowledge, many C-methyl flavonoids have been reported in the literature [15], [16], [17], but flavonoids possessing both C-methyl and C-prenyl groups are rarely reported. So far, only lespedezaflavanones F and G have been isolated from the root bark of Lespedeza formosa [18].

Compound 2 was obtained as a yellow powder with an [α] of − 5.9 (c 0.8, MeOH). HR‐ESI‐MS at m/z = 451.2129 [M – H]⁻ (calcd. for C₂₇H₃₁O₆: 451.2121) and EI‐MS at m/z = 452.4 [M]⁺ demonstrated the formula of 2 to be C₂₇H₃₂O₆. The IR spectrum showed absorptions of hydroxy and carbonyl groups at 3199 and 1631 cm⁻¹, respectively. The ¹H‐NMR spectrum ([Table 1]) of compound 2 displayed a flavanone skeleton by the resonances at δ _H = 5.91 (1H, br d, J = 12.6 Hz, H-2), 3.26 (1H, dd, J = 16.8, 12.6 Hz, H-3b), and 3.00 (1H, br d, J = 16.8 Hz, H-3a) [13]. The ¹³C‐NMR spectrum showed the corresponding carbon resonances at δ _C = 75.0 (C-2) and 43.2 (C-3). Two γ,γ-dimethylallyl groups were identified by the ¹H‐NMR spectral resonances at δ _H = 3.70 (4H, br s, H-1′′ and H-1′′′), δ _H = 5.68 (2H, br s, H-2′′ and H-2′′′), and δ _H = 1.70, 1.76, 1.80 × 2 (4 Me) and the related HMBCs. The HMBCs of H-2/C-9 (δ _C = 159.2) and H₂-1′′/C-7 (δ _C = 164.1), C-8 (δ _C = 108.8), and C-9 (δ _C = 159.2) revealed dimethylallyl and hydroxy groups located at C-8 and C-7, respectively. The other dimethylallyl group was placed at C-5′ owing to H-6′ (δ _H = 7.70) correlating with C-2 (δ _C = 75.0) and C-1′′′ (δ _C = 29.0) in the HMBC experiment. The assignments of Me-6, OH-5, MeO-2′, and OH-4′ were identified by HMBC analysis similar to that of 1. The (2S) absolute configuration was confirmed by its levorotatory optical activity and a negative Cotton effect at 290–300 nm in the CD spectrum [19], [20]. Based on the above evidence, 2 was established as (2S)-5,7,4′-trihydroxy-2′-methoxy-8,5′-di(3-methyl-2-butenyl)-6-methyflavanone.

Compounds 1 and 2 (purity > 99 %) were evaluated for their anti-inflammatory, antiproliferative, and antimicrobial activities. In the anti-inflammatory assay, compound 2 showed dose-dependent NO inhibitory effects, with an IC₅₀ value of 15.0 ± 0.7 µM compared with the positive control dexamethasone, which had an IC₅₀ value of 7.0 ± 1.8 µM; 1 did not show NO inhibitory effects. Both compounds showed cytotoxicity on RAW mouse macrophage cells starting from 25 µM. The two compounds displayed moderate antiproliferative activity against the tumor cell lines HT-29 and ZR-75-1, as shown in [Table 2]. In the antimicrobial assay, 1 and 2 did not show any inhibitive activity against Escherichia coli strain DH5-alpha.

Materials and Methods

Debarked stems and roots of T. wilfordii were purchased from Tian Heng drugstore (Beijing, P. R. China) and identified by Prof. De-an Guo. A voucher specimen (20080220) was deposited at the Department of Natural Medicine, School of Pharmaceutical Sciences, Peking University, Beijing, P. R. China.

The air-dried and ground stem and root material (9.5 kg) was extracted with 95 % EtOH. The ethyl acetate layer (135 g) of the 95 % EtOH extract was chromatographed on a silica gel column (1.5 kg, column: 100 × 10 cm) with gradient CH₃Cl−MeOH (100 : 0 to 50 : 50) elution to give 9 fractions. Compounds 1 and 2 were obtained from Fr. 2, together with friedelin, betulinic acid, β-amyrin, oleanolic acid, kaempferol, β-sitosterol, n-hexadecanoic acid, methyl hexadecanoate, n-octadecanoic acid, and n-tritriacontanoic acid (see details in Supporting Information).

Compound 1: Yellow needle-like crystal; m. p. 210–212 °C; [α]: 0 (c 0.3, MeOH); UV (MeOH): λ _max (log ε) = 272 (4.24) nm; IR (KBr): ν _max = 3387 (OH), 2932, 1643 (C=O), 1589, 1512, 1442 cm⁻¹; EI‐MS: m/z (rel. int.) = 382.4 [M]⁺ (30), 367.3 (55), 232.3 (7), 218.3 (13), 217.2 (100), 203.2 (15), 135.2 (10), 107.1 (12); HR‐ESI‐MS: m/z = 381.1335 [M – H]⁻ (calcd. for C₂₂H₂₁O₆: 381.1338); ¹H- and ¹³C‐NMR (pyridine-d ₅, 400/100 MHz) spectroscopic data: see [Table 1].

Compound 2: Yellow powder; m. p. 170–172 °C; [α]: − 5.9 (c 0.8, MeOH); UV (MeOH): λ _max (log ε) = 290 (4.10) nm; CD (c 0.02, MeOH): [θ] 298 = − 2.0; IR (KBr): ν _max = 3199 (OH), 2964, 2912, 1631 (C=O), 1596, 1517, 1447 cm⁻¹; EI‐MS: m/z (rel. int.) = 452.4 [M]⁺ (68), 397.4 (14), 248.3 (17), 235.3 (50), 219.3 (67), 205.3 (67), 191.2 (62), 179.2 (98), 163.2 (100), 91.1 (18), 83.2 (22), 69.2 (33), 55.2 (28), 43.2 (36); HR‐ESI‐MS: m/z = 451.2129 [M – H]⁻ (calcd. for C₂₇H₃₁O₆: 451.2121); ¹H- and ¹³ C‐NMR (pyridine-d ₅, 400/100 MHz) spectroscopic data: see [Table 1].

In vitro NO production inhibition assay [21]

The mouse macrophage cell suspension (100 µL) in DMEM was seeded in a 96-well plate at a concentration of 10⁵ cells per well and incubated overnight at 37 °C. Test compounds in various concentrations (1–25 µM) were added, and dexamethasone (97 %, D4902; obtained from Sigma) was used as a positive control. After 1 h, LPS (1 µg/mL) (L4516; obtained from Sigma) was added to wells as the triggering agent for NO induction. After 24 h incubation, NO concentrations were determined using a commercial Griess assay system (Promega). The cytotoxicity of compounds 1 and 2 on RAW mouse macrophage cells was measured through a cell proliferation assay by the MTS method [22].

In vitro antitumor cell line proliferation assay

The human breast cancer cell line ZR-75-1 and the human colon adenocarcinoma cell line HT were maintained in DMEM/F₁₂ (Dulbecco's modified Eagle's medium/Ham's nutrient mixture F-12) and DMEM (Invitrogen, Inc.) supplemented with 10 % (v/v) fetal bovine serum, penicillin G (100 U/mL), and streptomycin (100 µg/mL), respectively, and cultured in 96-well plates with 5 × 10⁴ cells per well. Appropriate dilutions of the test compounds 1 and 2 were added to the cultures, and 5-FU (99 %, 858 471; obtained from Aldrich) was used as a positive control. After 24 h, the survival rates of the cancer cells were evaluated by the MTS method [22] with the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega).

In vitro antimicrobial assay

The survival rates of the Escherichia coli strain DH5-alpha were tested by a broth dilution assay [23]. Details on this method are provided as Supporting Information.

X‐ray crystal structure analysis of 1

C₂₂H₂₂O₆, MW = 382.40, monoclinic, P2₁/c, a = 8.9776(4) Å, b = 15.9640(9) Å, c = 13.8468(7) Å, β = 108.134(3)°, V = 1885.93(17) ų, Z = 4. Suitable crystals of compound 1 for X-ray diffraction analysis were obtained from MeOH. A yellow needle-like crystal, approximate dimensions 0.10 × 0.12 × 0.50 mm, was mounted on a Bruker Apex II diffractometer with CuK α radiation (λ = 1.54 178 Å). The crystal was stabilized by an intermolecular hydrogen bond between O3 and O2 and by an intramolecular hydrogen bond between O4′ and O2 (− 1 + x, − 1 + y, 2/3 + z) (see Fig. 22S in Supporting Information). The graphics were prepared by PLATON [24]. Tables of crystallographic data have been deposited at the Cambridge Crystallographic Data Centre (reference number CCDC 758964). The supplementary crystallographic data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif.

Supporting information

General experimental procedures, extraction and isolation, spectral data (including ¹H‐and ¹³C‐NMR, DEPT, ¹H−¹H COSY, HMQC, HMBC, HR‐ESI‐MS, EI‐MS, UV, and IR spectra) of compounds 1 and 2, CD spectra of 2, hydrogen bond interactions in crystals of 1, the unit cell diagram of crystals of 1, X-ray crystal structure analysis, and in vitro biological activity assays are available as Supporting Information.

Acknowledgements

This work was supported by the program Changjiang Scholars and Innovation Team in University (No. 985–2–063–112) and by the Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China (No. 104218). The authors are grateful to Dr. Ilya Raskin, Reneta Pouleva, and Ph.D. candidate Brittany Graf for their bioassay assistance.

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1 These authors contributed to this article equally.

Prof. Dr. De-an Guo

School of Pharmaceutical Sciences
Peking University

38 Xueyuan Road

Beijing 100083

People's Republic of China

Phone: + 86 10 82 80 20 24

Fax: + 86 10 82 80 27 00

Email: gda@bjmu.edu.cn

References

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  CrossrefPubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
• 3 Ren J A, Tao Q S, Wang X B, Wang Z M, Li J H. Efficacy of T2 in active Crohn's disease: a prospective study report.  Dig Dis Sci. 2007;  52 1790-1797
  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 5 Brinker A M, Ma J, Lipsky P E, Raskin I. Medicinal chemistry and pharmacology of genus Tripterygium (Celastraceae).  Phytochemistry. 2007;  68 732-766
  CrossrefPubMedSearch in Google Scholar
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• 9 Seebacher W, Simic N, Weis R, Saf R, Kunert O. Complete assignments of ¹H and ¹³C NMR resonances of oleanolic acid, 18α-oleanolic acid, ursolic acid and their 11-oxoderivatives.  Magn Reson Chem. 2003;  41 636-638
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1 These authors contributed to this article equally.

Prof. Dr. De-an Guo

School of Pharmaceutical Sciences
Peking University

38 Xueyuan Road

Beijing 100083

People's Republic of China

Phone: + 86 10 82 80 20 24

Fax: + 86 10 82 80 27 00

Email: gda@bjmu.edu.cn

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