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Flavonolignans and Other Constituents from Lepidium meyenii with Activities in Anti-inflammatory and Human Cancer Cell Lines Naisheng Bai, Kan He, Marc Roller, Ching-Shu Lai, Lu Bai, and Min-Hsiung Pan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00219 • Publication Date (Web): 10 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Journal of Agricultural and Food Chemistry

Flavonolignans and Other Constituents from Lepidium meyenii with Activities in Anti-inflammatory and Human Cancer Cell Lines Naisheng Bai,† * Kan He, ‡ Marc Roller, ⊥ Ching-Shu Lai, § Lu Bai, † and Min-Hsiung Pan §

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†

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Engineering, Northwest University, Taibai North Road 229, Xi’an, Shaanxi, China 710069

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‡

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⊥

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§

Shaanxi Key Laboratory of Degradable Biomedical Materials, Department of Pharmacentical

Herbalife Interbational of America, 950 W. 190th Street, Torrance, CA 90502 Naturex SA, Site d’Agroparc BP 1218, 84911 Avignon Cedex 9, France

Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung, Taiwan

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*

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Telephone: 0086-29-88305682

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Fax: 0086-29-88302223

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E-mail: [email protected]

To whom correspondence should be addressed.

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ACS Paragon Plus Environment


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Abstract

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From the roots of Lepidium meyenii Walpers (Brassicaceae), two flavonolignans, tricin 4′-O-

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[threo-β-guaiacyl-(7′′-O-methyl)-glyceryl] ether (1) and tricin 4′-O-(erythro-β-guaiacyl-glyceryl)

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ether (2), along with eleven other known compounds,

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hydroxycinnamic acid (5), guanosine (6), glucotropaeolin (7), desulfoglucotropaeolin (8), 3-

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hydroxybenzylisothiocyanate (9), malic acid benzoate (10), 5-(hydroxymethyl)-2-furfural (11),

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D-phenylalanine (12), and vanillic acid 4-O-β-D-glucoside (13), have been isolated and

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identified.

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Structures were elucidated on the basis of NMR and MS data. Some isolates and previous

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isolated lepidiline B (14) were tested for cytotoxicity in a small panel of human cancer cell lines

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(Hep G2, COLO 205, and HL-60) and anti-inflammatory activities in LPS-treated RAW264.7

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macrophage. Among them, compound 1 and 14 were modestly active for inhibiting nitrite

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production in macrophage. Compounds 1, 14 and 3 were demonstrated to be selectively active

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against HL-60 cells with an IC50 of 40.4, 52.0, and 52.1 µM, respectively.

tricin (3), pinoresinol (4), 4-

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_____________________________________________________________________

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Keywords: Maca; Lepidium meyenii; Brassicaceae; flavonolignans, flavonoid, lignan,

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human cancer cell lines (Hep G2, COLO 205, and HL-60), anti-inflammatory

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INTRODUCTION

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Maca (Lepidium meyenii Walp.) is a well known crop in Peru with a long history and has been

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utilized in Andean region as a food and a medicine for centuries. As a food source, Maca

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displays a high nutritional value and is rich in sugars, proteins, starches and minerals.

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Medicinally, it has been used to enhance fertility, a property that tends to be reduced at high

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altitudes, both in humans and livestock.1 In recent years, scientific research in mice or rats has

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demonstrated some health benefits of maca, such as on improving sexual performance,2-4

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enhancing fertility,5 anti-fatigue,6 anti-depressant,7,8 spermatogensis,9 prevention of estrogen

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deficient bone loss,10 reducing prostate size in male rats with prostatic hyperplasia,11 balancing

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sex hormone levels,12 improving memory and learning,7,13 antioxidative activity, positive effect

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on the lipoprotein and triacylglycerol profile, and decreasing levels of glucose in blood.14

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Clinical studies in men showed that maca had a favorable effect on spermatogenesis15 and

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improved sexual desire,16 but did not affect serum reproductive hormone levels.17 Clinical data

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also indicated that supplementation of maca helped the perception of general and sexual well-

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being in young adult patients with mild erectile dysfunction.18 The effect of maca on alleviation

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of menopausal symptoms in early-postmenopausal women was reported in the double blind,

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randomized, placebo-controlled, and multi-centre clinical trials.19,20

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Although maca is a very popular dietary supplemental product in marketplace, not too many

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chemical constituents have been identified. Up to now, the major compounds are reported in

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maca include macaenes, macamides,21-23 glucosinolates,24,25 and alkaloids.22,25,26 We report

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herein the isolation and identification of two flavonolignans 1 and 2 along with 11 other known



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compounds from the roots of L. meyenii, as well as the biological evaluation of compounds 1-3

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and 14 for cytotoxicity (Hep G2, COLO 205, and HL-60) and anti-inflammatory activities in

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LPS-treated RAW364.7 macrophage.

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MATERIALS AND METHODS

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General Experimental Procedures. Optical rotations were measured with a Perkin-Elmer 241

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polarimeter.

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Instruments, Norwalk, CT). UV spectra were acquired on a Shimadzu, UV-1700 UV-Visible

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Spectrophotometer. The 1H and

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MHz) instrument (Varian Inc., Palo Alto, CA) with methanol-d4 (reference at 3.30 ppm) and

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DMSO-d6 as solvents (Aldrich Chemical Co., Allentown, PA). The 2D correlation spectra were

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obtained using standard gradient pulse sequences of Varian VNMR software and performed on

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4-nuclei PFG AutoSwitchable or PFG Indirect Detection probes. HRESIMS was run on Waters

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Micromass LCT (Waters Corporation, Milford, MA) or Thermo Scientific LTQ Orbitrap mass

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spectrometers (Thermo-Finnigan, San Jose, CA). ESI-MS were obtained on an LCQ ion trap

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HPLC analysis was performed on an Agilent 1100 LC Series using Luna C-8 column (5 micron,

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4.6 mm I.D. × 250, Phenomenex, Inc.) with a flow rate of 1.0 mL/min. Solvent system consisted

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of a linear gradient that started with 5 % (v/v) aqueous in 0.1% triflouroacetic acid and HPLC-

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grade acetonitrile and increased to 95% acetonitrile over 40 min, then increased to 100%

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acetonitrile over 5 min. At the end of the run, 100% of acetonitrile was allowed to flush the

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column for 5 min and an additional 10 min of post run time were set to allow for equilibration of

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the column with the starting eluant. UV detector was set at 280 nm wavelength and column

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temperature was ambient.

FT-IR was performed on a Perkin-Elmer spectrum BX system (PerkinElmer

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C NMR spectra were recorded on an Inova-400 (1H at 400


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Plant Material. The roots of maca, L. meyenii, were collected from the Andean Mountains of

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Peru. The species was identified using HPLC method by comparing with authentic sample. A

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voucher specimen (MA10205) was deposited in the Herbarium of Naturex, Inc.

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Extraction and Isolation of L. meyenii Constituents.

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Two different extracts were prepared before isolation. The first extraction was carried out with

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20 kg of maca root powder which was extracted by H2O three times (50 L×3) at room

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temperature. The extract was concentrated (4.3 kg) and chromatographed on the column of CG

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161 resin (Rohm and Haas, Philadelphia, PA) (27 L, 26 cm × 180 cm), eluting with H2O-EtOH

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(1:0, 9:1, 3:1, 2:1, 1:1, 0:1) solvent system. Volume of 3 L was used in each step and 1 L was

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collected as one fraction (named as I fractions). The 90% H2O-EtOH eluates (I 4-6, 78 g) were

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column chromatographed (CC) on a FPX 66 (Rohm and Haas, Inc.) (2.0 L, 12 cm × 50 cm) and

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eluted with H2O-EtOH (1:0, 9:1, 3:1, 2:1, 1:3, 0:1) solvent system. A 4.0-L solvent was used in

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each step and 1.0 L was collected as a fraction (named as II fractions). A total of 24 fractions

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were collected and concentrated. These fractions were separately and repeatedly subjected to CC

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over MCI GEL CHP-20P (Mitsubishi Kasei Co.) and/or Sephadex LH-20 (Sigma Chemical Co.,

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St. Louis, MO) (100 mL, 2.5 cm × 40 cm) and eluted with gradient H2O-MeOH solvent system

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to yield 6 (33 mg from fractions II 5-6, , tR = 6.0 min in HPLC), 7 (35 mg from II 5-6, tR = 9.8

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min), 8 (27 mg from II 7-9, tR = 10.3 min), 12 (25 mg from II 10-12, tR = 13.8 min), and 13 (5 mg

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from II 5-6, tR = 6.2 min). The 75% H2O-EtOH fractions (I 7-9, 22g) from CG 161 resin were

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CC on FPX 66 using the same condition as described above and produced III serial fractions.

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These fractions separately and repeatedly subjected to CC over MCI GEL CHP-20P and/or

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Sephadex LH-20 using gradient H2O-MeOH to yield 9 (16 mg from III 10-11, tR = 12.0 min), 10



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(156 mg from III 8-9, tR = 17.7 min), and 11 (162 mg from III 12-14, tR = 10.8 min). The second

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extraction was performed using 120 kg of maca roots. The air dried root powder was extracted

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with H2O twice (800 L x 2) at room temperature. After the H2O extract was drained, the

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remaining roots were extracted with 95% EtOH three times (800 L x 3). The EtOH solutions

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were combined and concentrated under reduced pressure to yield ethanol extract (6.1 kg) which

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was suspended in H2O and successively partitioned with hexane and EtOAc (10 L × 3). The

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EtOAc phase was concentrated (528 g) and CC on silica gel (2.5 kg, 12 cm × 50 cm), using a

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step gradient solvent system of hexane - acetone (10:1, 5:1, 2:1, 1:1, 0:1). In each step, a 5 L

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solvent was used and 1 L was collected as a fraction. Factions 12-18 were combined and

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concentrated (23 g) and were CC on polyamide resin (Sigma Chemical Co., St. Louis, MO) (500

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mL, 3.5 cm × 60 cm) using H2O-EtOH as solvent system (1:0, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, 0:1).

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A total of 1.5 L was used in each step and 0.5 L was collected as one fraction (named as IV

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fractions). These fractions were further CC over MCI GEL CHP-20P and/or Sephadex LH-20

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and eluted with gradient H2O-MeOH system to yield 1 (21 mg from IV 13-15, tR = 28.0 min), 2

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(17 mg from IV 9-12, tR = 25.1 min), 3 (2 mg from IV 9-12, tR = 28.5 min), 4 (3 mg from 9-12, tR

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= 13.9 min), and 5 (5 mg from IV 16-19, tR = 19.5 min).

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Tricin 4′-O-[threo- β-guaiacyl-(7′′-O-methyl)-glyceryl] ether (1): yellow amorphous powder;

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C28H28O11 ; [α]D25 –0.9º (c 1.00, MeOH); UV (MeOH) λmax (log ε) 215, 272, 340 nm; IR

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(amorphous powder) νmax 3316, 1595, 1419, 1182, 1025 cm-1; 1H and 13C NMR data, see Table

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1; HRESIMS m/z 541.1647 [M+H]+, (calculated for C28H29O11, 541.1639).

NMR and ESI-MS of Flavonoids.


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Tricin 4′-O-(erythro- β-guaiacyl-glyceryl) ether (2): yellow amorphous powder; C27H26O11 ;

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[α]D25 –0.9º (c 0.90, MeOH); UV (MeOH) λmax (log ε) 205, 270, 338 nm; 1H and 13C NMR data,

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see Table 1; HRESIMS m/z 527.1553 [M+H]+, (calculated for C27H27O11, 527.1542).

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Tricin (3): Yellow amorphous powder; C17H14O7; 1H NMR (400 MHz, CD3OD): δ 6.42 (1H, s,

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H-3), 6.18 (1H, d, J = 2.0 Hz, H-8), 5.98 (1H, d, J = 2.0 Hz, H-6), 7.18 (2H, s, H-2′& 6′H), 3.91

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(6H, s, 3′ & 5′-OCH3).

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163.1 (C-5), 100.2 (C-6), 165.8 (C-7), 95.2 (C-8), 159.3 (C-9), 104.5 (C-10), 122.5 (C-1′), 105.0

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(C-2′ & 6′), 149.5 (C-3′ & 5′), 141.0 (C-4′), 56.8 (C-3′ & 5′-OCH3). HRESIMS m/z 331.0811

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[M+H]+, (calculated for C17H15O7, 331.0818).

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Pinoresinol (4): White amorphous powder; C20H22O6; 1H NMR (400 MHz, CD3OD): δ 6.93 (2H,

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d, J = 1.2 Hz, H-2 $ 2′), 6.75 (2H, d, J = 8.0 Hz, H-5 & 5′), 6.80 (2H, dd, J = 8.0, 2.0 Hz, H-6 &

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6′), 4.69 (2H, d, J = 4.4 Hz, H-7 & 7′), 3.12 (2H, dd, J = 6.4, 4.4 Hz, H-8 & 8′), 4.21 (2H, dd, J =

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8.8, 6.8 Hz, H-9 & 9′), 3.81 (2H, dd, J = 8.8, 5.2 Hz, H-9 & 9′), 3.84 (6H, s, 3′ & 3′′-OCH3).

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NMR (100 MHz, CD3OD): δ 133.7 (C-1 & 1′), 110.9 (C-2 & 2′), 149.1 (C-3 & 3′), 147.3 (C-4

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& 4′), 116.0 (C-5 & 5′), 120.0 (C-6 & 6′), 87.5 (C-7 & 7′), 55.3 (C-8 & 8′), 72.6 (C-9 & 9′), 56.3

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(C-3′ & 3′′-OCH3). HRESIMS m/z 359.1489 [M+H]+, (calculated for C20H23O6, 359.1495).

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4-Hydroxycinnamic acid (5): White powder; C9H8O3; 1H NMR (400 MHz, CD3OD): δ 7.40 (2H,

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d, J = 8.8 Hz, H-2 & 6), 6.76 (2H, d, J = 8.8 Hz, H-3 & 5), 7.56(1H, d, J = 16.4 Hz, H-7), 6.24

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(1H, d, J = 16.4 Hz, H-8). 13C NMR (100 MHz, CD3OD): δ 127.1 (C-1), 131.1 (C-2 & 6), 116.8

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(C-3 & 5), 161.1 (C-4), 146.6 (C-7), 115.5 (C-8), 171.1 (C-9). ESI-MS (negative) m/z 163 [M-

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H]-, C9H7O3.

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C NMR (100 MHz, CD3OD): δ 166.1 (C-2), 104.3 (C-3), 183.7 (C-4),


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C


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Guanosine (6): White powder; C10H13N5O5; 1H NMR (400 MHz, DMSOd6): δ 7.93 (1H, s, H-

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8), 5.67 (H, d, J = 4.4 Hz, H-1′), 4.39 (1H, br. s, H-2′), 4.07 (1H, br. s, H-3′), 3.84 (1H, br. s, H-

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4′), 3.69 (1H, d, J = 11.2 Hz, H-5′a), 3.51 (1H, d, J = 11.2 Hz, H-5′b).

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DMSO-d6): δ 157.0 (C-1), 116.4 (C-2), 151.4 (C-3), 153.5 (C-5), 136.0 (C-8), 86.4 (C-1′), 73.6

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(C-2′), 70.3 (C-3′), 85.2 (C-4′), 61.8 (C-5′). ESI-MS (positive) m/z 284 [M+H]+, C10H14N5O5.

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C NMR (100 MHz,

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Glucotropaeolin (7): White powder; C14H18NO9S2K; 1H NMR (400 MHz, CD3OD): δ 7.38

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(2H, d, J = 7.6 Hz, H-2 & 6), 7.32 (2H, dd, J = 7.6, 0.8 Hz, H-3 & 5), 7.22 (1H, t, J = 7.2 Hz, H-

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4), 4.45 (H, d, J = 9.2 Hz, H-7a), 4.22 (H, d, J = 9.2 Hz, H-7b), 4.63 (H, br. s, H-1′), 4.03 (H, d, J

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= 16.0 Hz, H-6′a), 3.82 (H, d, J = 16.0 Hz, H-6′b), 3.57 (H, dd, J = 10.0, 2.4 Hz, H-4′), 3.07-3.23

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(3H, m, H-2′, 3′ & 5′).

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(C-3 & 5), 128.2 (C-4), 39.6 (C-7), 160.7 (C-8), 82.8 (C-1′), 74.1 (C-2′), 79.3 (C-3′), 71.1 (C-4′),

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82.2 (C-5′), 62.7 (C-6′). ESI-MS (negative) m/z 408 [M-H]-, C14H17NO9S2.

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C NMR (100 MHz, CD3OD): δ 137.4 (C-1), 129.3 (C-2 & 6), 129.9

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Desulfoglucotropaeolin (8): White powder; C14H19NO6S; 1H NMR (400 MHz, CD3OD): δ

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7.30 (2H, overlapped, H-2 & 6), 7.27 (2H, overlapped, H-3 & 5), 7.20 (1H, overlapped, H-4).

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39.0 (C-7), 153.4 (C-8), 82.1 (C-1′), 74.0 (C-2′), 79.0 (C-3′), 70.8 (C-4′), 81.7 (C-5′), 62.3 (C-6′).

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ESI-MS (negative) m/z 328 [M-H]-, C14H18NO6S.

C NMR (100 MHz, CD3OD): δ 138.0 (C-1), 128.8 (C-2 & 6), 129.4 (C-3 & 5), 127.6 (C-4),

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3-Hydroxybenzylisothiocyanate (9): White powder; C8H7NOS; 1H NMR (400 MHz, CD3OD):

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δ 6.72 (1H, s, H-2), 6.64 (1H, dd, J = 8.0, 2.8 Hz, H-4), 7.09 (1H, t, J = 8.0 Hz, H-5), 6.73 (H,

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dd, J = 8.0, 2.8 Hz, H-6), 3.40 (2H, s, H-7).

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C NMR (100 MHz, CD3OD): δ 138.2 (C-1),


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117.0 (C-2), 158.6 (C-3), 114.8 (C-4), 130.5 (C-5), 121.3 (C-6), 43.4 (C-7), 177.1 (C-8). ESI-MS

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(negative) m/z 163 [M-H]-, C8H6NOS.

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Malic acid benzoate (10): White powder; C11H10O6; 1H NMR (400 MHz, CD3OD): δ 7.97

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(2H, d, J = 8.0 Hz, H-2 & 6), 7.56 (2H, t, J = 7.2 Hz, H-3 & 5), 7.41 (1H, m, H-4), 5.61 (1H, dd,

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J = 7.2, 4.8 Hz, H-2′), 3.01 (2H, t, J = 4.0 Hz, H-3′).

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1), 130.6 (C-2 & 6), 134.7 (C-3 & 5), 129.5 (C-4), 167.1 (C-7), 173.1 (C-1′), 70.3 (C-2′), 36.9

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(C-3′), 172.4 (C-4′). ESI-MS (negative) m/z 237 [M-H]-, C11H9O6.

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C NMR (100 MHz, CD3OD): δ 130.2 (C-

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5-(Hydroxy-methyl)-furfural (11): Colorless oil; C6H6O3; 1H NMR (400 MHz, CD3OD): δ

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9.52 (1H, s, H-6), 7.38 (1H, d, J = 3.6 Hz, H-2), 6.57 (1H, d, J = 3.6 Hz, H-2), 4.60 (2H, s, H-5).

196

13

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ESI-MS (negative) m/z 125 [M-H]-, C6H5O3.

C NMR (100 MHz, CD3OD): δ 153.8 (C-1), 124.9 (C-2), 110.9 (C-3), 163.1 (C-4), 57.6 (C-5).

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D-Phenylalanine (12): White powder; C9H10 NO2; 1H NMR (400 MHz, CD3OD): δ 7.33 (5H,

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m, H-2, 3, 4, 5, & 6), 3.26 (H, dd, J = 15.2, 5.2 Hz, H-7a), 3.03 (H, dd, J = 15.2, 8.8 Hz, H-7b),

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3.85 (1H, dd, J = 8.8, 5.2 Hz, H-8). 13C NMR (100 MHz, CD3OD): δ 135.6 (C-1), 130.0 (C-2 &

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6), 130.4 (C-3 & 5), 128.7 (C-4), 37.2 (C-7), 55.5 (C-8), 171.8 (C-9). ESI-MS (negative) m/z 164

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[M-H]-, C9H10 NO2.

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Vanillic acid 4-O-β-D-glucoside (13): White powder; C14H18O9;

1

H NMR (400 MHz,

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CD3OD): δ 7.56 (1H, s, H-2), 7.16 (1H, d, J = 8.0 Hz, H-5), 7.60 (1H, d, J = 8.0 Hz, H-6), 3.86

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(3H, s, 3-OCH3), 5.00 (H, d, J = 7.2 Hz, H-1′), 3.52 (1H, m, H-2′), 3.40 (1H, m, H-3′), 3.48 (1H,

206

m, H-4′), 3.45 (1H, m, H-5′), 3.89 (1H, m, H-6′a), 3.67 (1H, dd, J = 12.0, 4.8 Hz, H-6′b).

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NMR (100 MHz, CD3OD): δ 126.1 (C-1), 114.1 (C-2), 150.1 (C-3), 151.8 (C-4), 116.1 (C-5),


13

C


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124.7 (C-6), 169.7 (C-7), 56.6 (3-OCH3), 101.8 (C-1′), 74.6 (C-2′), 77.7 (C-3′), 71.1 (C-4′), 78.2

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(C-5′), 62.3 (C-6′). ESI-MS (negative) m/z 329 [M-H]-, C14H17O9.

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Cell Culture and Chemicals. The COLO 205 cell lines were isolated from human colon

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adenocarcinoma (ATCC CCL-222). Human promyelocytic leukemia (HL-60) cells were

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obtained from American Type Culture Collection (Rockville, MD). The human HepG2

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hepatocellular carcinoma cell lines (BCRC 60025) were obtained from the Food Industry

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Research and Development Institute (Hsinchu, Taiwan). COLO-205 and HL-60 cell lines were

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grown at 37 oC in 5% CO2 atmosphere in RPMI. Hep G2 cells were grown in Dulbecco’s

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minimal essential medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum

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(Gibco BRL, Grand Island, NY), 100 units/mL of penicillin and 100 µg/mL of streptomycin, and

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kept at 37°C in a humidified atmosphere of 5% CO2 in air. Selected compounds were dissolved

219

in dimethyl sulfoxide (DMSO). Propidium iodide was obtained from Sigma Chemical Co. (St.

220

Louis, MO).

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Determination of Cell Viability. Cell viability was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-

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diphenyltetrazolium bromide (MTT) assay.15 Briefly, human cancer cells were plated at a density

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of 1×105 cells/mL into 24 well plates. After overnight growth, cells were pretreated with various

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concentration of test compounds for 24 h. The final concentration of dimethyl sulfoxide (DMSO)

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in the culture medium was < 0.05%. At the end of treatment, 30 µL of MTT was added, and the

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cells were incubated for a further 4 h. Cell viability was determined by scanning with an

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enzyme-linked immunosorbent assay reader with a 570 nm filter.


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Nitrite Assay. The RAW264.7 cells are treated selected compounds and LPS or LPS alone. The

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supernatants are harvested and the amount of nitrite, an indicator of NO synthesis, is measured

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by use of the Griess reaction. Briefly, supernatants (100 µL) are mixed with the same volume of

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Griess reagent (1% sulphanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine

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dihydrochloride in water) in duplicate on 96-well plates. After incubation at room temperature

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for 10 min, absorbance at 570 nm is measured with an ELISA reader (Thermo Labsystems

235

Multiskan Ascent, Kaikki Oikeudet, Finland).

236 237 238 239

RESULTS AND DISCUSSION

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provide two extracts. The separation was conducted by using a combination of column

241

chromatography on CG 161, FPX 66, silica gel, MCI GEL CHP-20P and Sephadex LH-20 and a

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total of thirteen compounds were isolated from the two extracts, including two flavonolignans,

243

tricin 4′-O-[threo- β-guaiacyl-(7′′-O-methyl)-glyceryl] ether (1)36 and tricin 4′-O-(erythro- β-

244

guaiacyl-glyceryl) ether (2),35 one flavonoid, tricin (3),28 together with pinoresinol (4),17 4-

245

hydroxycinnamic acid (5), guanosine (6), glucotropaeolin (7),25,30 desulfoglucotropaeolin (8),31

246

3-hydroxybenzylisothiocyanate (9),25,32 malic acid benzoate (10),25 5-(hydroxymethyl)-furfural

247

(11), D-phenylalanine (12), and vanillic acid 4-O-β-D-glucopyranoside (13).33 Compound 1, 2,

248

3, 5, 6, 8, 9, 11, and 13, were the first time to be isolated from this plant. All the structures were

249

established by spectroscopic methods, including

250

spectroscopy, MS, HRESIMS, and chemical properties. The known compounds were identified

The air-dried roots of L. meyenii were extracted with H2O and subsequently with 95% EtOH to

1

H,

13

C NMR, DEPT, 2D correlation



Page 12 of 23

251

by NMR data compared with those reported in the literature. Compounds 7, 8, 11, and 12 were

252

identified by direct comparison with commercial standards.

253 254

Compound 1 was obtained as a yellow amorphous powder. Its molecular formula C28H28O11 was

255

established based on the HRESIMS (m/z 541.1742 [M+H]+, calcd. for C28H29O11, 541.1710).

256

The IR spectrum showed the OH absorption (3316 cm-1) and aromatic rings (1615, 1595 cm-1).

257

UV spectrum of 1 exhibited maximum absorptions at 215, 272, 340 nm, typical for a flavone

258

derivative. The

259

conjugated ketonic carbon (δ 183.6), 20 aromatic or olefinic (δ 95.2-166.0), four methoxy (δ

260

56.3, 56.7, 56.7, 56.8), two oxygenated methine

261

methylene carbon signals (δ 61.7). These signals are corresponding to a methoxysand one

262

phenylpropanoid substituted flavonoid of 1. The 1H NMR spectrum contained signals of a 3′, 4′,

263

5′-trihydroxy substituted B-ring at δ 7.06 (2H, s, H-2′, 6′) and it was confirmed by the observed

264

long-range correlations of H-2′ (6′) with C-2 (δ 165.0), C-1′ (δ 127.4), C-3′ (5′ ) (δ 154.6), C-4′

265

(δ 140.2), and C-6′ (2′) (δ 104.7) in the gHMBC spectrum. The signal at δ 6.57 (1H, s, H-3) was

266

assigned to H-3 on the basis of its long-range correlations with C-2, C-4 (δ 183.6), C-10 (δ

267

105.5), and C-1′ in the gHMBC spectrum, as well as NOE correlation of H-3 with H-2′ (6′) in the

268

ROESY spectrum. Two broad singlets at δ 6.39 and δ 6.13 were assigned to be H-8 and H-6,

269

respectively, in the 5, 7-dihydroxy substituted A-ring. The assignments were confirmed by the

270

long-range correlations of H-8 with C-7 (δ 166.0), C-9 (δ 159.2), C-10 and H-6 with C-5 (δ

271

163.0), C-7, C-8 (δ 95.2), C-10 in the gHMBC spectrum, respectively. The correlations of two

272

O-methyl protons at δ 3.83 (6H, s, OCH3) with C-3′ (5′) (δ 154.6) in the gHMBC spectrum, as

273

well as the NOE correlations with H-2′ (6′) in the ROESY spectrum, indicated that two methoxy

274

groups were substituted at C-3′ and 5′.

13

C NMR spectrum revealed the presence of 28 carbon signals, including a

(δ 86.6 and 83.7), and one oxygenated

Thus, the flavone moiety was identified as 5, 7, 4′-


Page 13 of 23


275

trihydroxy-3′,5′-dimethoxyflavone (tricin). Additional NMR data showed the existing of a

276

guaiacylglycerol moiety by presence of an ABX spin system in 1H NMR at δ 6.75 (1H, br., s, H-

277

5′′), δ 6.80 (1H, dd, J = 4.8, 1.6 Hz, H-6′′), and δ 6.89 (1H, d, J = 1.6 Hz, H-2′′) for guaiacyl part

278

and signals at δ 4.46 (1H, d, J = 6.0 Hz, H-7′′), δ 4.43 (1H, m, H-8′′), δ 3.92 (1H, dd, J = 12.0,

279

4.4 Hz, H-9′′), and δ 3.73 (1H, dd, J = 12.0, 3.2 Hz, H-9′′) for glycerol part. The methoxy group

280

in guaiacyl was confirmed by the observed correlation between the O-methyl protons at δ 3.81

281

(3H, s, OCH3) and C-3′′(δ 148.8) in the gHMBC and NOE correlation between the methoxy

282

protons and H-2′′ in the ROESY spectrum, respectively. The fourth methoxy group (δ 3.89) (3H,

283

s, OCH3) was located at C-7″, since a 9.5 ppm downfield shift for C-7′′ (δ 83.7) in 1 was

284

observed, when compared to compound 2, where C-7′′ (δ 74.2) was hydroxyl group substituted

285

(34, 35). The linkage between the moieties of tricin and guaiacylglycerol was suggested through

286

4′-O-C-8″, as the signals of C-3′, 5′ (δ 154.6) of 1 showed 5.1 ppm downfield shift comparing

287

with those of tricin (3) at C-3′, 5′ (δ 149.5). Very similar chemical shifts of C-8″ and C-9″ of 1

288

to those found in 2 and literature data34 were in accord with the above suggestion. The coupling

289

constant of JH-7′′,H-8′′ for 1 was 6.0 Hz in methanol-d6, corresponding to a threo form of chiral

290

centres at C-7″ and C-8″ based on empirical observation.35,36 Therefore, 1 was identified to be

291

tricin 4′-O-[threo- β-guaiacyl-(7′′-O-methyl)-glyceryl] ether, and 2 was idenfied to be tricin 4′-O-

292

(erythro-β-guaiacyl-glyceryl) ether, two flavonolignan compounds from L. meyenii.” The

293

completed 1H and 13C NMR assignments are given in Table 1.

294 295

Compounds 1, 2, 3 and lepidiline B (14, previously isolated from Lepidium meyenii)26 were

296

tested with regard to their effects on nitrite production in LPS-activated macrophages. As shown

297

in Table 2, when RAW264.7 cells were treated with test compounds at 40 µg/mL and LPS (100



298

ng/mL), the potency of inhibitory effect on nitrite production showed the sequence as 1 > 14 > 3

299

> 2 where 1 and 14 significantly inhibited the NO production. The four compounds were also

300

assayed for cytotoxicity in HL-60, Hep G2, and COLO 205 cell lines. Compounds 1, 3, and 14

301

showed moderate to weak effect against HL-60 cells with IC50 of 40.4, 52.1, and 52.0 µM,

302

respectively, while no cytotoxicity were observed for these compounds in Hep G2 and COLO

303

205 cell lines (Table 3). Compound 14 was previously reported against the UMUC3, PACA2,

304

MDA231, and FDIGROV cell lines with ED50 values of 6.47, 1.38, 1.66, and 5.26 µg/mL,

305

respectively.25 The three isolated flavonoids in the present investigation are all consisting of a

306

tricin unit. Tricin which exists as glycoside in rice bran and other grass species has been

307

considered as a potential cancer chemopreventive agent in humans.37 Some other isolated

308

compounds in the current study have also been reported to possess different biological activities.

309 310 311

ACKNOWLEDGEMENTS

312

The authors wish to thank Naturex Inc. for funding this project. The authors wish to thank Dr.

313

Baoning Su, Wuxi Pharma Inc., for the collaboration on the HRESIMS analyses and valuable

314

suggestions for the chemical structural identification.

315 316

REFERENCES

317

1. Johns, T. The anu and the maca. J. Ethnobiol. 1981, 1, 208-212.

318

2. Zheng, B. L.; He, K.; Kim, C. H.; Rogers, L.; Shao, Y.; Huang, Z. Y.; Lu, Y.; Yan, S.

319

J.; Qien, L. C.; Zheng, Q. Y. Effect of a lipidic extract from Lepidium meyenii on

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sexual behavior in mice and rats. Urology 2000, 55, 598−602.


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3. Cicero, A. F.; Bandieri, E; Arletti, R. Lepidium meyenii Walp. improves sexual

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behaviour in male rats independently from its action on spontaneous locomotor

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activity. J. Ethnopharmacol. 2001, 75, 225–229.

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4. Cicero, A. F. G.; Piacente, S.; Plaza, A.; Sala, E.; Arletti, R.; Pizza, C. Hexanic Maca

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extract improves rat sexual performance more effectively than methanolic and

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chloroformic Maca extracts. Andrologia 2002, 34, 177–179.

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5. Ruiz-Luna, A.C.; Salazar, S.; Aspajo, N.J.; Rubio, J.; Gasco, M,; Gonzales, G. F.

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Lepidium meyenii (Maca) increases litter size in normal adult female mice. Reprod.

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Biol. Endocrinol. 2005, 3: 16.

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6. Zheng, B.L.; He, K.; Hwang, Z.Y.; Lu, Y.; Yan, S. J.; Kim, C. H.; Zheng, Q. Y.,

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Effect of aqueous extract from Lepidium meyenii on mouse behavior in forced

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swimming test, In Quality Management of Nutraceuticals; Ho, C. T., Zheng, Q. Y.,

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7. López-Fando1, A.; Gómez-Serranillos, M. P.; Iglesias, I.; Lock, O.; Upamayta, U. P.;

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Carretero, M. E. Lepidium peruvianum chacon restores homeostasis impaired by

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8. Rubio, J.; Caldas, M.; Dávila, S.; Gasco, M.; Gonzales, G.F. Effect of three different

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cultivars of Lepidium meyenii (Maca) on learning and depression in ovariectomized

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mice. BMC Complementary and Altern. Med. 2006, 6(23), 1-7.

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9. Gonzales, G. F.; Gasco, M.; Córdova, A.; Chung, A.; Rubio, J.; Villegas, L. Effect of

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Lepidium meyenii (Maca) on spermatogenesis in male rats acutely exposed to high

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altitude (4340 m). J. Endocrinol. 2004, 180, 87–95.



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10. Zhang, Y.; Yu, L.; Ao, M.; Jin, W. Effect of ethanol extract of Lepidium meyenii

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Walp. on osteoporosis in ovariectomized rat, J. Ethnopharmacol. 2006, 105, 274–279.

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11. Gonzales, G. F.; Vasquez, V.; Rodriguez, D.; Maldonado, C.; Mormontoy, J.; Portella,

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J.; Pajuelo, M.; Villegas, L.; Gasco1, M. Effect of two different extracts of red maca

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in male rats with testosterone-induced prostatic hyperplasia. Asian J. Androl, 2007, 9,

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245–251.

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12. Meissner, H. O.; Mrozikiewicz, P.; Bobkiewicz-Kozlowska, T.; Mscisz, A.; Kedzia,

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B.; Lowicka, A.; Reich-Bilinska, H.; Kapczynski, W.; Barchia, I.; Hormone-

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balancing effect of pre-gelatinized organic maca (Lepidium peruvianum Chacon): (I)

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Biochemical and pharmacodynamic study on maca using clinical laboratory model on

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ovariectomized rats. Int. J. Biomed. Sci. 2006, 2, 260-272.

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13. Rubio, J.; Qiong, W.; Liu, X.; Jiang, Z.; Dang, H.; Chen, S.-L.; Gonzales, G. F.

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Aqueous extract of Black Maca (Lepidium meyenii) on memory impairment induced

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by ovariectomy in mice. Evidence- Based Complementary and Alternative Medicine,

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14. Vecera, R.; Orolin, J.; Skottová, N.; Kazdová, L.; Oliyarnik, O.; Ulrichová, J.;

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Simánek, V. The influence of maca (Lepidium meyenii) on antioxidant status, lipid

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and glucose metabolism in rat. Plant Foods Hum. Nutr. 2007, 62, 59–63.

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15. Gonzales, G. F.; Cordova, A.; Gonzales, C.; Chung, A.; Vega, K.; Villena, A.

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Lepidium meyenii (maca) improved semen parameters in adult men. Asian J. Androl.

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2001, 3, 301-303.


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16. Gonzales, G. F.; Córdova, A.; Vega, K.; Chung, A.; Villena, A.; Góñez, C.; Castillo,

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S.; Effect of Lepidium meyenii (MACA) on sexual desire and its absent relationship

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with serum testosterone levels in adult healthy men. Andrologia 2002, 34, 367-372.

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17. Gonzales, G. F.; Córdova, A.; Vega, K.; Chung, A.; Villena, A.; Góñez, C. Effect of

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Lepidium meyenii (Maca), a root with aphrodisiac and fertility-enhancing properties,

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on serum reproductive hormone levels in adult healthy men. J. Endocrinol. 2003, 176,

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18. Zenico1, T.; Cicero, A. F.; Valmorri1, L.; Mercuriali, M.; Bercovich, E. Subjective

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effects of Lepidium meyenii (Maca) extract on well-being and sexual performances in

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patients with mild erectile dysfunction: a randomised, double-blind clinical trial.

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Andrologia 2009, 41, 95–99.

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19. Meissner, H. O.; Mscisz, A.; Reich-Bilinska, H.; Kapczynski, W.; Mrozikiewicz, P.;

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Bobkiewicz-Kozlowska, T.; Kedzia, B.; Lowicka, A.; Barchia, I. Hormone-balancing

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effect of pre-gelatinized organic maca (Lepidium peruvianum Chacon): (II)

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Physiological and symptomatic responses of early-postmenopausal women to

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standardized doses of maca in double blind, randomized, placebo-controlled, multi-

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centre clinical study. Int. J. Biomed. Sci. 2006, 2, 360-374.

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20. Meissner, H. O.; Mscisz, A.; Reich-Bilinska, H.; Mrozikiewicz, P.; Bobkiewicz-

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Kozlowska, T.; Kedzia, B.; Lowicka, A.; Barchia, I. Hormone-balancing effect of

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pre-gelatinized organic maca (Lepidium peruvianum Chacon): (III) Clinical responses

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of early-postmenopausal women to maca in double blind, randomized, placebo-

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controlled, crossover configuration, outpatient study. Int. J. Biomed. Sci. 2006, 2,

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21. Zheng, B. L.; Kim, C. H.; Wolthoff, S.; He, K.; Rogers, L.; Shao, Y.; Zheng, Q. Y. U.S. Patent 6,267,995, 2001. 22. Muhammad, I.; Zhao, J.; Dunbar, D. C.; Khan, I. A. Constituents of Lepidium meyenii ‘maca’. Phytochemistry 2002, 59, 105−110. 23. Zhao, J.; Muhammad, I.; Dunbar, D. C.; Mustafa, J.; Khan, I. A. New alkamides from Maca (Lepidium meyenii). J. Agric. Food Chem. 2005, 53, 690–693. 24. Dini, I.; Tenore, G. C.; Dini, A. Glucosinolates from Maca (Lepidium meyenii). Biochem. Sys. Ecol. 2002, 30, 1087–1090.

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25. Piacente, S.; Carbone, V.; Plaza, A.; Zampelli, A.; Pizza, C. Investigation of the tuber

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constituents of Maca (Lepidium meyenii Walp.). J. Agric. Food Chem. 2002, 50,

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5621-5625.

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26. Cui, B. L.; Zheng, B. L.; He, K.; Zheng, Q. Y. Imidazole alkaloids from Lepidium meyenii. J. Nat. Prod., 2003, 66, 1101–1103.

401

27. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application

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to proliferation and cytotoxicity assays. J. Immunol. Methods. 1983，65, 55–63.

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28. Kuwabara, H.; Mouri, K.; Otsuka, H.; Kasai, R.; Yamasaki, K. Tricin from a

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Malagasy Connaraceous plant with potent antihistaminic activity. J. Nat. Prod. 2003,

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66, 1273-1275.

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29. Carpinella, M. C.; Giorda, L. M.; Ferrayoli, C. G.; Palacios, S. M. Antifungal effects

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of different organic extracts from Melia azedarach L. on phytopathogenic fungi and

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their isolated active components. J. Agric. Food Chem. 2003, 51, 2506-2511.

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30. Prestera, T.; Fahey, J. W.; Holtzclaw, W. D.; Abeygunawardana, C.; Kachinski, J. L.; Talalay, P. Comprehensive chromatographic and spectroscopic methods for the


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separation and identification of intact glucosinolates. Anal. Biochem. 1996, 239,

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31. Kiddle, G.; Bennett, R. N.; Botting, N. P.; Davidson, N. E.; Robertson, A. A.;

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Wallsgrove, R. M. High - performance liquid chromatographic separation of natural

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and synthetic desulphoglucosinolates and their chemical validation by UV, NMR and

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chemical ionization - MS methods. Phytochem. Anal. 2001, 12, 226–242.

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32. Fahey, J. W.; Zalcmann, A. T.; Talalay, P. The chemical diversity and distribution of

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glucosinolates and isothiocyanates among plants. Phytochemistry 2001, 56, 5−51.

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33. Sakushima, A.; Coskun, M.; Maoka, T. Hydroxybenzoic acids from Boreava orientalis. Phytochemistry 1995, 40, 257-261. 34. Bouaziz, M.; Veitch, N. C.; Grayer, R. J.; Simmonds, M. S. J.; Damak, M.

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Flavonolignans from Hyparrhenia hirta. Phytochemistry 2002, 60, 515−520.

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35. Nakajima, Y.; Yun, Y. S.; Kunugi, A. Six new flavonolignans from Sasa veitchii

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(Carr.) Rehder. Tetrahedron 2003, 59, 8011−8015.

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36. Jung, L.H.; Lee, D.Y.; Cho, J.G.; Lee, S.M.; Kang, H.C.; Seo, W.D.; Kang, H.W.;

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Kim, J.Y.; Baek, N.I. A new flavonolignan from the aerial parts of Oryza sativa L.

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inhibits nitric oxide production in RAW 264.7 macrophage cells. J. Korean Soc. Appl.

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Chem. 2011, 54, 865-870.

429

37. Verschoyle, R. D., Greaves, P., Cai, H., Borkhardt, A., Broggini, M., D’Incalci, M.,

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Gescher, A. J.. Preliminary safety evaluation of the putative cancer chemopreventive

431

agent tricin, a naturally occurring flavone. Cancer Chemotherapy and Pharmacology,

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2006, 57(1), 1-6.

433



434 435 436

Table 1. 1H, 13C NMR and HMBC Data for 1 and 2 (CD3OD)a No. 1 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 3′,5′OCH3 1′′ 2′′ 3′′ 4′′ 5′′ 6′′ 7′′

δΗ

1 δC

6.57s

165.0s 105.6d

6.13s 6.39s

7.06s

7.06s 3.83s

6.89 br. s

6.75 br. s 6.80m 4.46d(6.0)

3′′OCH3 7′′OCH3

HMBC (H to C)

183.6s 163.0s 100.2d 166.0s 95.2d 159.2s 105.5s 127.4s 104.7d 154.6s 140.2s 154.6s 104.7d 56.7q 131.0s 112.0d 148.8 147.2 115.6d 122.0 83.7d

threo form 8′′ 9′′

437 438 439 440 441 442 443 444 445 446 447 448 449

Page 20 of 23

4.43m 3.92dd(12.0, 4.4) 3.73dd(12.0,3.2) 3.81s

56.3q

3.89s

56.8q

2,4,10,1′ ROE: 2′,6′

5,7,8,10 7,9,10

2,1′,3′,4′,6′

2,1′,2′,4′,5′ 3′ or 5′ ROE: 2′,6′ 3′′,4′′,6′′

1′′, 2′′,3′′ 1′′, 3′′,4′′,7′′ 1′′, 2′′,6′′,8′′, 9′′ , 7′′-OCH3

86.6d 61.7t

No. 1 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 3′,5′OCH3 1′′ 2′′ 3′′ 4′′ 5′′ 6′′ 7′′

2 δC

6.56s

165.0s 105.6d

6.12s 6.36s

7.07s

7.07s 3.85s

6.97 d(1.6)

6.72d(8.0) 6.79dd(8.0,1.6) 4.91d(4.4)

183.7s 163.1s 100.2d 166.1s 95.2d 159.2s 105.4s 127.5s 104.8d 154.7s 140.4s 154.7s 104.8d 56.8q 133.7s 111.5d 148.6s 146.9 115.6d 120.8d 74.2d

erythro form 8′′ 9′′

3′′ ROE: 2′′

δΗ

3′′OCH3

4.41m 3.89dd(12.0, 4.4) 3.65dd(12.0,3.2) 3.80s

a

HMBC (H to C)

2,4,10,1′ ROE: 2′,6′

5,8,10,9′′ 3,4,7,9,10,9′′

2,1′,3′,4′,6′

2,1′,2′,4′,5′ 3′ or 5′ ROE: 2′,6′,7′′,8′′ 3′′,4′′,6′′,7′′

1′′, 2′′,3′′,4′′,7′′ 2′′,6′′,8′′,7′′OCH3,

87.5d 61.9t 56.4q

3′′ ROE: 2′′

Carbon multiplicities were determined by DEPT experiments (s = C, d= CH, t = CH2, q = CH3); Figures in parentheses denote J values (Hz).


Page 21 of 23

450 451 452 453

454 455 456 457

467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486


Table 2. Effect of 1, 2, 3, and 14 on LPS-induced nitrite production in RAW 264.7 macrophage. 1 2 3 14 Control 0.3 ± 0.6 0.3 ± 0.6 0.3 ± 0.6 0.3 ± 0.6 LPS 27.2 ± 3.0 27.2 ± 3.0 27.2 ± 3.0 27.2 ± 3.0 12.5 ± 1.2 24.2 ± 1.2 21.9 ± 1.9 6.3 ± 2.2 20 µg/mL 1.7 ± 0.4 24.5 ± 2.3 16.8 ± 0.6 4.3 ± 1.9 40 µg/mL

Table 3. Effect of different compounds on the growth of various human cancer cells. 458 459 Compound Cell line HL-60 Hep G2 COLO 205 460 IC50 (µM) 40.4 ± 1.9 >100 >100 461 1 >100 >100 >100 462 2 52.1 ± 3.7 >100 >100 463 3 52.0 ± 1.1 >100 >100 464 14 a) 4.64±0.05 4.82±0.05 16.12±1.49465 Doxorubicin 466 Cells were treated with various concentrations of selected compounds for 24 h. Cell viability then was determined by the MTT assay as the described. Each experiment was independently performed three times and expressed as mean ± SE. a) Positive control compound.


R O

3

H C O

3

H C O

3

3

H C O

H C O

O

O H H O

' 4

O

H O

' ' 3

' ' 7

' 3 H ' O 9H C O

O

O H

O H O

O H O

3333

e M = R 1

H O

O

H = R 2

H N

N

2

O

H O H O

O H

6666

4444

H O O C

l C

N + N

H O

3

3 1

4 1

HH OO2 H C

O O H O H C O


H N N N O O H

3

H C O

O

O 3 H C

487 488 489 490 491 492 493 494

Page 22 of 23 Journal of Agricultural and Food Chemistry

Page 23 of 23


Flavonolignans and Other Constituents from Lepidium meyenii with Activities in Anti-inflammatory and Human Cancer Cell Lines

Naisheng Bai,† * Kan He, ‡ Marc Roller,  Ching-Shu Lai, §Lu Bai, † and Min-Hsiung Pan §


Flavonolignans and Other Constituents from ... - ACS Publications

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