A new anti-inflammatory β-carboline alkaloid from the hairy-root cultures of Eurycoma longifolia

Abstract One new β-carboline alkaloid 7-methoxy-(9H-β-carbolin-1-il)-(E)-1-propenoic acid (1) together with 9-methoxycanthin-6-one (2) and 9-hydroxycanthin-6-one (3) were isolated from the hairy-root cultures of Eurycoma longifolia. The effects of these compounds on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells were investigated. Compound 1 strongly inhibited the production of NO while 2 and 3 having weak or inactive effect. Consistently, compound 1 decreased the expression of cyclooxygenase-2 and inducible nitric oxide synthase. Graphical abstract


Introduction
Eurycoma longifolia Jack (Simaroubaceae) is an important medicinal plant in South-East Asian countries due to its broad pharmaceutical properties such as anti-inflammation, anticancer, antidiabetes and antimalaria (Bhat & Karim 2010). The roots of this plant have been widely used as a single ingredient or as a part of a mixture with other herbs for the treatment of sexual insufficiency, malaria, aches, dysentery and fever. In Vietnam, E. longifolia is named 'cay ba benh' , meaning a tree that cures hundreds of diseases (Loi 2004). Previous studies have reported that the plant contains quassinoids, canthin-6-one and β-carboline alkaloids, squalene derivatives, tirucallane-type triterpenes and biphenylneolignans (Kuo et al. 2003;Park et al. 2014;Tran et al. 2014). However, E. longifolia roots are harvested after several years of cultivation, i.e. time-consuming and seasonal depending, and the uprooting harvest for medicinal preparation has caused an adverse effect on the natural population of E. longifolia. Thus, an alternative method of E. longifolia production is required to meet the ever-growing demand of this medicine. To solve this problem, the use of plant tissue culture to produce high-value secondary compounds for pharmaceutical and industrial purposes is ever increasing. In comparison with different plant tissues, hairy-root cultures have been reported many advantages such as continuous growth periods, rapid growth rates, easy culturing in hormone-free media and accessibility to genetic manipulation and thus is a useful approach for the production of active compound in confined and secure environments. Hairy roots can be obtained by infecting the wounded higher plants with Agrobacterium rhizogenes, a gram negative soil bacterium that can transfer a DNA segment (T-DNA) from root-inducing (Ri) plasmid into the genome of the infected plant. This T-DNA carries a set of genes that encode enzymes which control auxin and cytokinin biosynthesis, which induces the formation of hairy roots (Guillon et al. 2006). In this study, cultured hairy roots of E. longifolia were produced using the A. rhizogenes-mediated transformation system. A chromatographic fractionation of 90 g dried mass of hairy roots led to the isolation of a new β-carboline alkaloid (1) together with 9-methoxycanthin-6-one (2) and 9-hydroxycanthin-6-one (3) (Kardono et al. 1991).

Results and discussion
Compound 1 was obtained as amorphous yellow powder. Its HR-ESI-MS spectrum revealed the peak at m/z 269.0929 [M+H] + corresponding to the molecular formula C 15 H 13 N 2 O 3 . The 1 H-NMR spectrum of 1 showed the signals characteristic of an ABX aromatic system at δ H 6.95 ( . The 13 C-NMR and DEPT spectra of 1 indicated the presence of a methoxy, seven methines and seven quaternary carbons. A carboxylic (δ C 166.8) and two methine (δ C 128.9 and 129.1) signals were attributed for a propenoic acid moiety. The rest of signals corresponded to the β-carboline skeleton previously found in Eurycoma sp. (Kuo et al. 2003;Park et al. 2014;Tran et al. 2014). These data were very similar to those of 6-methoxy-(9H-β-carbolin-1-il)-(Z)-2-propenoic acid as previously reported (Nunomura et al. 2012). However, the large coupling constant (J = 13.0 Hz) of the olefinic protons H-1′ and 2′ indicated the trans double bond in 1. Extensive analysis of HMBC correlations ( Figure 1) confirmed that the methoxy group attached to C-7. Thus, 1 was elucidated to be 7-methoxy-(9H-β-carbolin-1-il)-(E)-1-propenoic acid.
All of the isolated compounds were tested for their inhibition of NO production in LPSstimulated RAW264.7 cells. As a result, the β-carboline 1 strongly prevented the NO production in a dose-dependent manner while two canthin-6-ones 2 and 3 were weak or inactive up to 30 μM ( Figure 2). The cell viability assay indicated that the inhibition of NO production was not resulted from cytotoxic effects of the tested compounds (data not shown). It is known that cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are two major inflammatory mediators, and the induced expression of iNOS is primarily responsible for the NO production. Thus, the effects of 1 on LPS-induced expressions of COX-2 and iNOS in RAW264.7 cells were also investigated using Western blot analysis. As the result, 1 dose-dependently suppressed the LPS-induced expressions of iNOS and COX-2 proteins. It has been reported that β-carboline alkaloids exert extensive biochemical activities and multiple pharmacological effects such as anti-inflammation, antitumor, antibacteria and antithrombosis (Lee et al. 2000;Cao et al. 2007;Zhang & Sun 2015). Consistently, our results showed that the β-carboline 7-methoxy-(9H-β-carbolin-1-il)-(E)-1-propenoic acid (1) strongly down-regulated the inflammatory iNOS and COX-2 expression.
In conclusion, this work identifies a new anti-inflammatory β-carboline, 7-methoxy-(9Hβ-carbolin-1-il)-(E)-1-propenoic acid, from the cultured hairy roots of E. longifolia. This compound showed strong inhibitory effect on the NO production and decreased COX-2 and iNOS protein expression in LPS-induced RAW264.7 cells. Further studies are in progress to determine the mode of action of this anti-inflammatory agent.

General procedures
TLC was performed using precoated Kiesel gel 60 F254 and visualised by UV light 254 nm and 10% H 2 SO 4 reagent with heat. Column chromatography was performed using silica gel 60 (Merck, 70-230 mesh). NMR experiments were carried out on a Bruker AM500 FT-NMR spectrometer using tetramethylsilane as internal standard. The HR-ESI-MS spectra were recorded on an API Q-STAR PULSAR I of Applied Biosystem.

Plant materials
The seeds of E. longifolia Jack were collected in July 2011 in Bai Tu Long National Park, Quang Ninh, Vietnam, and identified by Dr Bui Van Thanh, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology. The voucher specimens (BTL11) were deposited at the Institute of Biotechnology.

Preparation of cultured hairy roots
Seeds of E. longifolia Jack were obtained from a mature tree in Bai Tu Long National Park, Quang Ninh province, Vietnam. Seeds were washed thoroughly with detergent and rinsed under running tap water for 5 min. After removing the seed coat, the seeds were sterilised with 70% ethanol for 1 min and 0.1% mercuric chloride solution for 3 min, followed by rinsing with autoclaved water. Sterilised seeds were germinated on MS medium supplemented with 3% sucrose and GA3 (0.3 mg/L). Cotyledons and hypocotyls from 3-week-old in vitro cultures were used for the A. rhizogenes transformation.
A colony of A. rhizogenes strain ATTC 15834 (provided by Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Germany) was cultured on 50 mL YMB medium at 28 °C, 200 rpm on an orbital shaker for 14-16 h. Then, the bacterial cells were collected by centrifugation at 5000 rpm for 10 min and resuspended in 25 mL ½ Woody Plant (WP) medium. Cotyledons and hypocotyls were cut in 0.3-0.4 cm in ½ WP liquid medium. Explants dried on sterile filter papers were transferred onto a new plate containing 25 mL of agrobacterial suspension and incubated for 30 min with gentle shaking. The bacteria were poured off, and the explants were dried on sterile filter paper and placed on WP solid medium for 2 days under low light intensity. After three days inoculated with bacteria, the explants were placed onto new WP plates containing 500 mg/L cefotaxime (Brithol Michcoma, Holland). The explants were subcultured on new WP antibiotic plates every two weeks until hairy root tips appeared. Hairy root tips (4-5 cm in length) were then separately transferred to 50-mL flasks containing 10 mL of WP liquid medium and placed on a shaker at 60 rpm, 25 ± 2 °C for further analysis. A hairy root exerting high growth rate was selected to produce hairy root biomass. Four grams of hairy roots biomass were cultured in 150 mL liquid WP medium (semi-submerged culture) at 25 °C and orbitally shaked 60-70 rpm in the dark. After 3 weeks of cultures, 200 mL of WP fresh medium was supplied into the cultures. After 6 cultured weeks, the biomass obtained was dried at 37 °C and average production of hairy root (5 g dry weight, DW). Hairy root was continuously cultured for high amount of mass, and the dry mass was used for next experiments.

Extraction and isolation
The cultured hairy roots of E. longifolia (90 g dried mass) were extracted with methanol in a sonic bath (500 mL × 3 times). The combined extracts were concentrated to give 13.5 g crude extract which was then partitioned between 10% HCl solution and ethyl acetate (each 500 mL). After the removal of organic layer, the acidic solution was adjusted to pH 10 by 1 N NaOH and then extracted with ethyl acetate (300 mL × 3 times) to obtain an alkaloid residue (651 mg). The alkaloid extract was chromatographed on a silica gel column eluted with dichloromethane-methanol (25:1 v/v) to afford 12 fractions F1-12. Compound 3 (15.0 mg) was recrystallised in dichloromethane from F4. Compound 2 (96.0 mg) was purified from F2 by a C18 column using methanol-water 3:1 (v/v). Fraction F12 was chromatographed on a silica gel column eluted with dichloromethane-methanol (20:1 v/v) to obtain 1 (29.6 mg).

Assay for inhibition of NO production and iNOS, COX-2 expression
Determination of NO production and the immunoblot analysis for iNOS and COX-2 were performed as previously described (Trang et al. 2014). Celastrol was used as a positive control for the NO assay (IC 50 , 1.0 μM).