key: cord-0895807-1x0bb513
authors: TANG, Wen-Zhao; WANG, Ying-Ai; GAO, Tian-Yang; WANG, Xiao-Jing; ZHAO, Yun-Xue
title: Identification of C-geranylated flavonoids from Paulownia catalpifolia Gong Tong fruits by HPLC-DAD-ESI-MS/MS and their anti-aging effects on 2BS cells induced by H2O2
date: 2017-05-31
journal: Chinese Journal of Natural Medicines
DOI: 10.1016/s1875-5364(17)30059-6
sha: 4a0245e89f0e0e1c3ed5dac4dc5793c761b99e87
doc_id: 895807
cord_uid: 1x0bb513

Abstract The fruits of Paulownia catalpifolia Gong Tong are used as a Chinese folk herbal medicine for the treatment of enteritis, tonsillitis, bronchitis, and dysentery, etc. Our previous study has identified new C-geranylated flavanones with obvious anti-proliferative effects in lung cancer A549 cells. In the present study, a new C-geranylated flavone, paucatalinone C (1) and five known C-geranylated flavanones (2–6) were isolated. In addition, a total of 34 C-geranylated flavonoids were detected by HPLC-DAD-ESI-MS/MS coupling techniques from the CH2Cl2 extract of P. catalpifolia. Futhermore, anti-aging effects of isolated compounds were evaluated in vitro with premature senescent 2BS cells induced by H2O2. Phytochemical results indicated that P. catalpifolia was a natural resource of abundant C-geranylated flavonoids. Diplacone (3) and paucatalinone A (5) were the potent anti-aging agents in the premature senescent 2BS cells induced by H2O2 and the C-geranyl substituent may be an important factor because of its lipophilic character.

Fruits of the genus Paulownia plants (Scrophulariaceae family), also named as "Pao Tong Guo" in China, are used as a Chinese folk herbal medicine for the treatment of enteritis, tonsillitis, bronchitis, and dysentery, etc [1] . Previous phytochemical studies on Paulownia plants have led to the isolation of quinones [2] , lignans [3] , phenylpropanoid glycosides [4] , and C-geranylated flavonoids [5] [6] [7] [8] [9] . Up to date, more than 40 C-geranylated flavanones have been isolated from Paulownia plants, especially from P. tomentosa [10] . Some C-geranylated flavanones display cytotoxic [7, 11] , antioxidant [5, 11] , antiviral [12] , antibacterial [6, 9] , anti-inflammatory [13] , and anti-cholinesterase activities [14] . Therefore, C-geranylated flavanones have drawn great attentions because of their various prominent pharmacological effects.

The fruits of P. catalpifolia Gong Tong are also used as a Chinese folk herbal medicine. However, few phytochemical and pharmacological studies have been carried out on this plant so far. Previously, three new C-geranylated flavanones have been obtained from its fruits [15] , which reveal obvious cytotoxic activity and structural novelty, encouraging us to carry out the continuous research. In the present study, another new C-geranylated flavone, paucatalinone C (1), along with other five known C-geranyl flavanones were isolated from the fruits. In addition, using HPLC-DAD-ESI-MS/MS coupling techniques, more than 34 C-geranylated flavonoids were detected. Furthermore, the cellular anti-aging effects of all isolated C-geranylated flavonoids on the premature senescent human embryonic lung diploid fibroblasts cells (2BS cells) induced by H 2 O 2 were investigated. Our phytochemical results indicated that P. catalpifolia was also a natural resource of abundant C-geranylated flavonoids and paucatalinone C (1) was the first isolation from the genus Paulownia as a C-geranylated flavone. Biological activity assays demonstrated diplacone (3) and paucatalinone A (5) were the potent anti-aging agents and the C-geranyl substituent may be an important factor for the cellular bioactive effects because of its lipophilic character.

The IR spectra were recorded as KBr disks on a Nicolet Impact 410 FT-IR spectrophotometer. The NMR spectra were obtained on Bruker Avance DPX 400 MHz spectrometers. The ESIMS were measured on an Agilent 1100 Series LC/MSD trap mass spectrometer. The HPLC system was consisted of a binary pump (G1312 A), an autosampler (G1313 A), a degasser (G1322 A), and DAD (G1315 B) controlled by software (v. A08.03). Separations were achieved on an Agilent SB-C 18 column (RP-18, 250 mm × 4.6 mm; 5 μm). The mass detector was an ion trap spectrometer (G2445A) equipped with an electrospray ionization system (ESI). The HRESIMS were measured on Bruker FTMS APEXIII 7.0T mass spectrometer. Column chromatography was performed on ODS (40−70 μm), and Sephadex LH-20. Prep-HPLC was carried out on a Shimadzu LC-6AD with an SPD-10A detector. A reversed-phase C 18 column (YMC Pack ODS-A 20 mm × 250 mm, 10 μm) was employed.

In the present study, mature fruits of P. catalpifolia Gong Tong were collected in October 2012 from Shengfuo Mount, Yiyuan, China and authenticated by Prof. XIN Yi-Zhou, Shandong University of Traditional Chinese Medicine. A voucher specimen (No. 20121003) was deposited in the herbarium of Institute of Materia Medica, Shandong Academy of Medical Sciences. The collected fruits were dried in shade and broken to powder.

The dried fruits powder (1.5 kg) was extracted with 95% aqueous EtOH (total 8 L) thrice. It was refluxed for 2 h each time. The combined solution was filtered and concentrated under reduced pressure to afford the ethanolic extract (120 g). The majority of the ethanolic extract was suspended with water. After defatted by cyclohexane, the residue was extracted successively with CH 2 Cl 2 and n-BuOH. The yields of the CH 2 Cl 2 and n-BuOH fractions were about 21 and 53 g, respectively.

The samples were analyzed using an Agilent HPLC 1100

Series instrumente quipped with a diode array detector and a mass detector in series (Agilent Technologies, Waldbronn, Germany). The HPLC mobile phase was HPLC-grade acetonitrile (solvent A) and water (containing 0.1% acetic acid, V/V, solvent B) at flow rate of 1.0 mL·min −1 . Elution was performed with a gradient starting with 5% A, to reach 20% A at 20 min, 45% A at 50 min, 58% A at 70 min, and 80% A at 90 min, and then became isocratic for 5 min. UV chromatograms were recorded at 290 and 340 nm. The mass nebulizer gas was nitrogen. The pressure and the flow rate of the dryer gas were set at 65 psi and 11 L·min −1 , respectively. The heated capillary and voltage were maintained at 350 °C and 4 kV, respectively. Mass scan (MS) and daughter (MS-MS) spectra were measured from m/z 100 to 1 000. Collision-induced fragmentation experiments were performed in the ion trap using helium as the collision gas, with voltage ramping cycles from 0.3 to 2 V. Mass spectrometry data were acquired in the positive mode.

The isolation of C-geranylated flavonoids from CH 2 Cl 2 fraction was guided by HPLC-DAD-ESI-MS/MS, and accomplished by ODS, Sephadex LH-20 column chromatography and prep-HPLC. CH 2 Cl 2 fraction (5.0 g) was separated by Sephadex LH-20 with 80% aqueous MeOH as eluent to yield three subfractions. The second subfraction (3.6 g) with little green pigmentum was performed a HPLC-DAD-ESI-MS/MS assay firstly, then was chromatographed on an ODS column, eluting with aqueous MeOH from 40% to 100% in gradient to yield six subfractions (Sub. F 1-6). Each subfractions was then executed an analysis by HPLC-DAD-ESI-MS/MS for the presence of C-geranylated flavonoids. Sub. F 4 (820 mg) gave a distinct signal with typical features of C-geranylated flavone (1) and was purified again by prep-HPLC with 82% aqueous MeOH as mobile phase to yield the new compound (8.6 mg, t R = 32 min). Sub. F 3 was chromatographed on ODS eluting with 65% aqueous MeOH and purified by Sephadex LH-20 with MeOH to give compound 2 (10.3 mg). Compound 3 (20.0 mg) was isolated from sub.F 5 by ODS chromatography with 75% aqueous MeOH as elution. Compounds 4-6 were obtained in our previous study [15] and were detected by HPLC-DAD-ESI-MS/MS in the present study.

The basic structural characteristics of the new compound were deduced from its UV, IR, and NMR spectra. The structures of known compounds were elucidated by comparing their 1 H and 13 C NMR spectral data with those reported as 3′, 4′, 5, 7-tetra-hydroxy-6-[7-hydroxy-3, 7-dimethyl-2(E)-octenyl] flavanone (2) [16] and diplacone(3) [17] . 

The human embryonic lung diploid fibroblasts cells (2BS cells) were originally established at the National Institute of Biological Products (Beijing, China). The 2BS cells were considered to be young at PD30 or below and cultured in Dulbecco's modified Eagle medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin G/streptomycin sulfate (GIBCO). Different test compounds were diluted to 0.01, 0.10, and 1.00 µmol·L −1 for this assay and eriodictyol and luteolin were used as reference compounds. Cell viability was assessed by the MTT-staining assay as we described previously [18] . Each compound was tested in triplicate and the experiments were repeated three times.

A HPLC signal (Fig. 5a, 1 , t R = 68.18 min) possessed the distinctive characteristics of geranylated flavone. It was then isolated by column chromatography and prep-HPLC under HPLC-DAD-ESI-MS/MS analysis.

Compound 1 (Fig. 1, 1 ) was isolated as a yellow powder and its positive HCl-Mg reaction and UV data ( max 240 (sh), 275, and 352 nm) indubitably indicated that it was a flavonoid. Its molecular formula, C 26 H 28 O 7 , was demonstrated on HRESIMS and NMR spectroscopic data (Table 1 ). In its 1 H NMR spectrum, two aromatic proton singlets at δ 6.53 and 2 ). In addition, another three methyl singlets at δ H 1.57, 1.59 and 1.79, three methylenes at δ H 1.97, 2.06 and 3.33 in addition to two olefinic protons at δ 5.07 (t, H-6"), 5.25 (t, H-2") were attributed to a geranyl substituent, which was also certified by HMBC experiment (Fig. 2) . HMBC correlations of H-1" at δ 3.33 with C-6 (δ 113.4), C-5 (δ C 160.1), and C-7 (δ C 163.8) indicated that the geranyl group was attached to C-6 of ring A (Fig. 2) . According to the above analyses, compound 1 was assigned to be 6-geranyl-4', 5', 5, 7-tetrahydroxy-3'methoxyflavone, named as paucatalinone C.

C-geranylated flavonoids are a kind of natural compounds that combine a flavonoid skeleton with a lipophilic geranyl side-chain. C-geranylated flavones/flavonols and flavanones/ flavanonols can be distinctly distinguished by their basic UV and ESI-MS/MS characteristics. Generally, Flavones or flavonols showed two main maximum UV absorptions at about λ 220−280 and 300−400 nm, respectively; however, flavanones or flavanonols showed a main maximum absorption at about λ 290 nm with a shoulder peak at about λ 230 nm and a weak absorption at about λ 340 nm. Besides, the key ESI-MS/MS characteristic of geranylated flavonoids was a neutral loss of a C 9 unit from the C 10 side chain and the appearance of the flavonoid skeleton fragment with a methylene ion [19] [20] . These neutral C 9 units and methylene flavonoid skeletons can The neutral loss Δm/z 124 was easily considered as the C 9 unit U1 fragmented from the side chain (Fig.  5b) . So 1 was demonstrated as a geranyl flavone or flavonol. Based on its NMR, structure of 1 was elucidated completely and confirmed the deduction of UV and MS (Figs. 1 and 2) . HPLC signal 4 (Fig. 5a , t R = 44.7 min) was paucatalinone B, a geranyl flavanone with an oxidated geranyl substituent at C-6 isolated from this plant previously [15] . Its UV spectra were detected at  max 228 (sh), 295 (main), and 342 nm and ESI-MS showed a neutral loss Δm/z 142 from m/z 473 [M + H] + in MS 1 to m/z 331 [M + H] + in MS 2 , which indicated that the C 9 unit fragment was U3 and the methylene flavonoid skeleton was F5 (Fig. 5c) . These UV and MS information were completely satisfied to a geranyl flavanone.

Genus Paulownia is a rich natural resource of abundant C-geranylated flavonoids, especially for P. tomentosa [10] . Isolated Paulownia C-geranylated flavonoids display structural diversity. Up to date, except paucatalinone C (1) reported in this paper as a geranyl flavone, all of them were flavanones or flavanonols. The C 10 side-chain can be the geranyl group, also be further modified by hydroxylation, etc. Main neutral losses with Δm/z of different C 9 unit from the C 10 side chain in MS 2 are summarized in Fig. 3 . The location of C 10 side-chain is almost all attached at C-6 in A-ring, except for paucatalinone A (5) and isopaucatalinone B (6) at C-8 isolated from P. catalpifolia Gong Tong [15] . In addition, most of the isolated C-geranylated flavanones are characterized by a 2S configuration in contrast to the dihydroflavonols, for which 2R, 3R isomer is often observed. Furthermore, methoxy groups are always located at C-3′ or C-3′, 5′ in B-ring of the flavonoid skeleton. Based on the above analysis, possible methylene flavonoid skeletons are subjectively summarized in Fig. 4 with the methylene group at C-6 as representative.

On the basis of HPLC-DAD-ESI-MS/MS experiments, more than 34 C-geranylated flavonoids were detected from the CH 2 Cl 2 extract. Their UV spectra, quasi-molecular mass, and MS/MS data are presented in Table 2 were also indicated in this plant as its microconstituents. Previously, two C-pentenyl flavanonols have been isolated from P. tomentosa [5, 8] , of which 6-isopentenyl-3′-O-methyltaxifolin must be 23 with the same UV and MS information [5] . Our previous phytochemical investigation made it clear that structures of geranylated flavonoids in P. catalpifolia Gong Tong were different from to those in other plants of the same genus. The difference was mainly due to the isolation of the C-8 substituted geranyl flavanones [15] along with the detection and isolation of C-geranylated flavones/ flavonols in this experiment. Unfortunately, accurate structures for each detected HPLC signal could not be demonstrated definitely only by UV and MS experiments because of the above uncertainties. Results of this analytical assay only gave us information that abundant geranylated flavonoids were found in fruits of P. catalpifolia Gong Tong with their structural type (flavanones or flavones), even potential structures. If intensive phytochemical isolations were carried out for this kind of compounds, especially for those microconstituents, many new C-geranylated flavonoids with slight structural changes could be obtained and elucidated.

To compare the biological properties of these isolated components, cell viability was estimated using an MTT assay on premature senescent 2BS cells induced by H 2 O 2 . As a result, all the isolated components demonstrated better cellular bioactivities than those of positive controls and the parent flavonoids luteolin and eriodictyol. Young 2BS cells can be caused premature senescence induced by H 2 O 2 for a short time and it has long been used as a model system for anti-aging induction [21] . In the present anti-aging assay, obvious growth stimulation by isolated C-geranylated flavonoids was found in a dose-dependent manner, with 1.0 µmol·L −1 showing maximal effect. Briefly, it was found that an exposure of 2 BS cells to 100 µmol·L −1 of H 2 O 2 for 20 h caused a significant livability decrease of approximately 32.8%. After treatment with 0.10 and 1.0 µmol·L −1 of different C-geranylated flavonoids solutions and incubation with H 2 O 2 to induce oxidative stress, the protective effects of these solutions on cell survival were all significantly increased than those of positive control Vitamin C and their parent flavonoids luteolinor and eriodictyol. Furthermore, diplacone (3) and paucatalinone A (5) revealed more effective anti-aging activity with the increasing cell livability than those of the other four compounds, 1, 2, 4, and 6 ( Table 3 ).

The bioactivities revealed that the C 10 side chain at position C-6 or C-8 perhaps was an important factor, Table 2 especially for the geranyl substituent with no modifications (1, 3, 5). If the geranyl moiety modified by hydroxylation at C-7" (2, 4, 6) , the bioactivities could be significantly decreased, which was particularly verified by the cellular radical-scavenging assay. This is probably due to the lipophilic character of the geranyl group. It can increase the alteration of membrane fluidity for tested compounds and keep 2BS cell from cell membrane and nuclear damage when the cells are exposed to an oxidative or genotoxic stress. 

P. catalpifolia Gong Tong is a natural resource of abundant C-geranylated flavonoids with structural variety as its typical bioactive constituents. Because of the structural novelty and various prominent pharmacological effects of C-geranylated flavonoids, it is necessary to carry out future investigations of new components and different pharmacological activities of extracts from P. catalpifolia Gong Tong.

Jiangsu New Medical College. The Chinese Medicine Dictionaty

Shanghai People's Publishing House

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