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NF-κB Inhibitors from Eurycoma longifolia

The roots of Eurycoma longifolia have been used in many countries of Southeast Asia to alleviate various diseases including malaria, dysentery, sexual insufficiency, and rheumatism. Although numerous studies have reported the pharmacological properties of E. longifolia, the mode of action of the anti-inflammatory activity has not been elucidated. Bioguided isolation of NF-κB inhibitors using an NF-κB-driven luciferase reporter gene assay led to the identification of a new quassinoid, eurycomalide C (1), together with 27 known compounds including 11 quassinoids (2–12), six alkaloids (13–18), two coumarins (19, 20), a squalene derivative (21), a triterpenoid (22), and six phenolic compounds (23–28) from the extract of E. longifolia. Evaluation of the biological activity revealed that C19-type and C20-type quassinoids, β-carboline, and canthin-6-one alkaloids are potent NF-κB inhibitors, with IC50 values in the low micromolar range, while C18-type quassinoids, phenolic compounds, coumarins, the squalene derivative, and the triterpenoid turned out to be inactive when tested at a concentration of 30 μM. Eurycomalactone (2), 14,15β-dihydroklaieanone (7), and 13,21-dehydroeurycomanone (10) were identified as potent NF-κB inhibitors with IC50 values of less than 1 μM.


■ RESULTS AND DISCUSSION
The methanolic root extract of E. longifolia was separated by liquid−liquid extraction with water and solvents of increasing polarity (n-hexane, diethyl ether, ethyl acetate, and n-butanol). The fractions obtained were dried, redissolved in DMSO, and assayed for their ability to inhibit the TNF-α-induced activation of NF-κB. HEK-293/NF-κB-luc cells were treated with test materials at a concentration of 10 μg/mL (positive control: parthenolide 5 μM) and stimulated with TNF-α. Cell viability was determined simultaneously by CellTracker Green CMFDA staining. 15 The diethyl ether and ethyl acetate fractions showed a significant NF-κB inhibitory activity and no significant cytotoxicity (see Figure S1, Supporting Information). Thus, these fractions were separated by Sephadex LH-20 and silica gel column chromatography as well as fast centrifugal partition chromatography to give a new quassinoid, eurycomalide C (1), and 27 known compounds (2−28).
Among these compounds, quassinoids and alkaloids were found as the main components in the active fractions. It is interesting that a chromone derivative (isoaleoresin D, 27), which so far has been reported only in the genus Aloe, was identified also as a constituent of E. longifolia. In addition, a triterpenoid (pedunculoside, 22) and a flavonoid (3,5,6,7,8,3′,4′-heptamethoxyflavone, 28) were isolated for the first time from this plant.
Quassinoids are known for cytotoxicity 35 against some cell lines (CaOv-3, HeLa, HepG2, HM3KO, MCF-7), while others (MDBK, Vero) seem to be not affected. Accordingly, the isolated quassinoids were investigated for their effects on cell viability. At a concentration of 30 μM, none of the tested  (Table S1, Supporting Information) showed growth inhibitory activity against HEK-293/NF-κB-luc cells. This is in line with recent studies that investigated the acute toxicity of a diethyl ether fraction of E. longifolia and some of its constituents in a mouse model. After oral application, the LD 50 value of the diethyl ether fraction was 2.31 g/kg body weight, while one of the isolated quassinoids, eurycomanone (9), showed an LD 50 value of 122.5 μM/kg (0.05 g/kg) body weight. 36 The same study evaluated also effects in a brine shrimp toxicity assay, affording LD 50 values of 144.8, 323.5, 3.5, and 10.3 μg/mL for compounds 6, 7, 9, and 10, respectively. Interestingly, the acute toxicity-guided fractionation afforded only quassinoids of the C 20 -type (7−10), while other types [the C 18 -type (11 and 12), the C 19 -type (1−6)] were not detected. A recent clinical study using a standardized water-soluble extract of E. longifolia (Physta) containing 0.8−1.5% eurycomanone (9) (200 mg twice a day) did not reveal adverse effects. 37 From this it can be concluded that discrepancies in cytotoxicity data of quassinoids are likely due to the different cell model used and varying assay conditions. However, under the particular experimental conditions used in the present study the isolated compounds had no cytotoxic effects.

Journal of Natural Products
Among the isolated alkaloids, 1-methoxycarbonyl-β-carboline (13) exhibited weak NF-κB effects, with an IC 50 value of 29.3 μM. In a previous study, another β-carboline alkaloid (4,8dimethoxy-1-vinyl-β-carboline) isolated from Melia azedarach L. var. japonica was reported to suppress NO synthesis in LPS/ interferon γ-activated RAW 264.7 cells through the inhibition of iNOS protein expression due to decreased mRNA transcription. 37 Moreover, the mechanism of this effect was explained by the capability of β-carboline alkaloids to suppress the NF-κB signaling pathway through inhibition of IKK activity in LPS-stimulated RAW 264.7 macrophages. 38 Although the content of compound 13 in E. longifolia roots is only low (based on the isolated amount), it might contribute to the inhibitory NF-κB effect of the extracts.
The main constituents of the active extracts of E. longifolia were identified as quassinoids belonging to the C 18 -type (11 and 12), C 19 -type (1−6), and C 20 -type (7−10). It is interesting that C 18 -type quassinoids did not show inhibitory activity, while C 19 -and C 20 -type quassinoids were found to be responsible for the effect of the extracts that exhibited NF-κB inhibition in the low μM range (IC 50 values from 0.5 to 18.4 μM). Preliminary structure−activity relationship data could be established for this compound class. First, since the cyclopentenone in ring A of C 18 -type quassinoids abrogated this effect, the six-membered ring A is clearly necessary for such activity. Second, the double bond between carbons 3 and 4 seems to be more relevant than the double bond between carbons 5 and 6 (compound 1 gave a higher IC 50 value (>30 times) than compound 2). Third, exchange of one of the hydroxy groups at C-2 or C-7 by a carbonyl functionality resulted in a significant decrease of NF-κB inhibition (compound 2, IC 50 0.5 μM; compound 3, IC 50 = 1.5 μM; compound 5, IC 50 = 3.8 μM).
In conclusion, the present results demonstrated that the methanolic extract of the roots of E. longifolia has potent NF-κB inhibitory effects. Investigation of the active fractions led to identification of basically two main compound classes responsible for the activity: (i) C 19 -and C 20 -type quassinoids and (ii) β-carboline and canthin-6-one alkaloids. The observed bioactivity of the isolated compounds might be a first indication for their molecular mode of action and will help to elucidate the traditional use of the root of E. longifolia against inflammation. The study resulted in the discovery of canthin-6-one alkaloids as a new compound class acting as NF-κB inhibitors.
The Eury-DE fraction (5.5 g) was subjected to Sephadex LH-20 CC, eluted with MeOH to obtain 11 fractions (EuryA1 to EuryA11). Fraction EuryA9 was applied to silica gel CC (DCM/MeOH, 99.5:0.5 to 98.0:2.0, v/v) to afford compound 15 (4.6 mg). Fraction EuryA9-12 (29.8  The noncrystalline residue of fraction EuryA7 was applied to FCPC (n-hexane/CHCl 3 /MeOH/H 2 O, 1:3:3:2, lower phase: mobile phase) to afford 10 fractions (EuryA7-R1 to EuryA7-R10). Fraction EuryA7-fluorescent probe is retained inside living cells, it can be used to monitor cell membrane integrity and has been widely used to quantify viable cells. 15,39 Afterward, cells were reseeded in 96-well plates (4 × 10 4 cells/well) in phenol red-free and FBS-free DMEM overnight. Cells were then pretreated with the investigated samples or with solvent vehicle (0.1% DMSO in culture medium) for 30 min and stimulated with TNF-α (2 ng/mL) for 4 h. Then, cells were lysed in a luciferase lysis buffer (Promega; E1531), and the luminescence of the firefly luciferase and the fluorescence of the CellTracker Green CMFDA were quantified with a Genios Pro plate reader (Tecan, Grodig, Austria). For quantification of NF-κB activity, the luciferasederived signal from the NF-κB reporter was normalized by the CellTracker Green CMFDA-derived fluorescence to account for differences in cell number. Potential differences in cell viability were detected by comparison of the CellTracker Green CMFDA fluorescence of the solvent vehicle treated cells and cells treated with the indicated samples.
Statistical Analyses. Nonlinear regression (with sigmoidal dose response) was used to calculate the IC 50 values using GraphPad Prism 4.03 (GraphPad Software, Inc.). Statistical differences were compared with ANOVA analysis followed by Tukeys' test. A p-value < 0.01 was considered significant.