Identification of β-carboline and canthinone alkaloids as anti-inflammatory agents but with different inhibitory profile on the expression of iNOS and COX-2 in lipopolysaccharide-activated RAW 264.7 macrophages
- 558 Downloads
A compound library, which consists of 75 natural β-carboline-type or canthinone-type alkaloids from Simaroubaceae plants and their chemical synthetic analogues, was screened for the anti-inflammatory activity by inhibition of the overproduction of inflammatory mediator nitric oxide (NO) in lipopolysaccharide (LPS)-activated RAW 264.7 macrophage cells. Six compounds, namely, benzalharman (23), kumujian (27), 1-ethyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (37), 1-acetophenone-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (42), cathin-6-one (46), and 9-methoxy-cathin-6-one (57), exhibited significant inhibitory activity on the overproduction of NO with good dose dependency. Further investigation demonstrated that all of the six compounds down-regulated the high expression of inducible nitric oxide synthase (iNOS) protein. Among them, two canthinone-type alkaloids (46 and 57) potently down-regulated cyclooxygenase-2 (COX-2) protein expression in a dose-dependent manner and also inhibited the overproduction of inflammatory mediator prostaglandin E2 (PGE2). However, the β-carboline-type alkaloids (23, 27, 37, and 42) exhibited no obvious inhibition on the overproduction of PGE2 and the expression of COX-2 protein. The results suggested that β-carboline-type alkaloids and canthinone-type alkaloids may exert an anti-inflammatory effect through different mechanism.
Keywordsβ-Carboline alkaloid Canthinone alkaloid Anti-inflammatory activity NO PGE2 iNOS COX-2
Nitric oxide (NO) is a biological messenger molecule and neurotransmitter, which is synthesized by NO synthase (NOS) in multiple cells. Three types of NOS, namely neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), produce NO through the catalytic reaction of l-arginine and dioxygen. Different from nNOS and eNOS, iNOS is expressed only after being induced by extracellular stimuli such as stimulation with lipopolysaccharide (LPS) . In addition, prostaglandin E2 (PGE2) is the main metabolite of arachidonic acid epoxy synthase, which is important for cell growth or regulation and can cause some symptoms of inflammation, such as swelling, redness, and heat . In mammal cells, two types of cyclooxygenase (COX-1 and COX-2) have been identified . COX-2, the key enzyme in the process of PGE2 synthesis, is highly expressed during the process of inflammatory reaction induced by LPS . Many studies have reported that the high expression of iNOS and COX-2 promotes the overproduction of NO and PGE2 in activated macrophages, respectively. Excessive production of such inflammatory mediators and high expression of inflammatory proteins can result in chronic inflammatory diseases [5, 6, 7]. Chronic inflammation has also been linked with various diseases, such as systemic lupus erythematosus, rheumatoid arthritis, hepatitis, cancer, and so on [8, 9, 10, 11].
β-Carboline-type alkaloids and canthinone-type alkaloids represent the major bioactive constituents of medicinal plants belonging to the Simaroubaceae family, and a large range of bioactivities have been reported due to the diversity of chemical structure [12, 13, 14, 15, 16]. For example, β-carboline-1-propionic acid and 1-hydroxy-canthin-6-one from Ailanthus altissima have been reported to possess potent inhibitory activity against cyclic adenosine monophosphate phosphodiesterase . 4,5-Dimethoxy-10-hydroxy-canthin-6-one, canthin-6-one, 8-hydroxy-canthin-6-one, 4,5-dimethoxy-canthin-6-one, and 5-hydroxy-4-methoxy-canthin-6-one from Picrasma quassioides have been reported as having cytotoxic activity against human nasopharyngeal carcinoma (CNE2) cells .
During our continuing search for bioactive compounds from natural resources, we have established a compound library, which consists of alkaloids from Simaroubaceae plants, such as Picrasma quassioides, Picrasma javanica, Ailanthus altissima, Simarouba amara, Eurycoma longifolia, Simaba cuspidata, and Quassia amara, as well as their chemical synthetic analogues. The chemical structures are shown in the Supplementary data, which included 38 β-carboline alkaloids (7–45), 31 canthinone alkaloids (1, 3–6, and 46–70), and 6 dimeric alkaloids (71–76). Previous bioassay of this compound library has led to the discovery of a number of protein tyrosine phosphatase-1B (PTP1B) inhibitors  and the cerebral protective agents . In the present study, the compound library was assayed for anti-inflammatory activity by inhibiting the overproduction of the inflammatory mediator NO in LPS-activated RAW 264.7 macrophage cells, which is a classical in vitro inflammatory cell model. Furthermore, six selected bioactive compounds were further investigated for their inhibitory effect on PGE2 production and the modulatory mechanism on the high expression of iNOS and COX-2 proteins.
Results and discussion
The inhibitory activity on the overproduction of NO was firstly assessed at a final concentration of 100 μM for all compounds. The level of nitrite as an indicator of NO was determined by the Griess reaction, and the cell viability was investigated by the MTT assay. Hydrocortisone sodium succinate (HSS, Tianjin Biochem Pharmaceutical Co., Ltd.), a clinically commonly used anti-inflammatory drug, was used as the positive control in this bioassay. Among the 75 tested compounds, 14 (11–13, 15, 21–23, 27, 29, 30, 31, 37, 41, and 42) out of 38 β-carboline-type alkaloids showed potent NO inhibitory activity (the inhibitory rate on NO production was higher than 80%). Among them, seven compounds (12, 13, 15, 22, 23, 27, and 42) exhibited cytotoxicity (cell viability less than 50%). However, these 7 compounds did not show cytotoxicity when at a lower concentration of 50 μM. On the other hand, five compounds (46, 47, 55, 57, and 64) out of 31 canthinone-type alkaloids showed potent NO inhibitory activity without cytotoxicity. Among the six dimeric alkaloids, three compounds (71, 72, and 76) inhibited the NO production, but also showed cytotoxicity (Supplementary data). The results indicated the potential of a number of β-carboline-type alkaloids and canthinone-type alkaloids in the compound library as anti-inflammatory agents. Compounds (21, 46, and 57) have been reported with inhibitory NO production activities [21, 22, 23], but compounds (11–13, 15, 22, 23, 27, 29–31, 37, 41, 42, 47, 55, 64, 71, 72, and 76) were not reported as inhibitors of NO production.
The bioactive β-carboline-type alkaloids can be divided into three types, namely, type I, with an ethyl moiety at C-1 (11, 12, and 13); type II, with a double bond at C-1′ (methoxycarbonyl: 22, 27, 29, 30, and 31; carbon–carbon double bond: 21 and 23); and type III, with a saturated C ring and a methoxy-carbonyl at C-3 (37, 41, and 42). Compound 37 showed the strongest NO inhibitory activity, which suggest that the ethyl group at C-1 may be more contributive than other groups at the C ring. Among canthinone-type alkaloids, compounds (46, 47, 57, and 64) without substituent groups at C-4 and C-5, were far more active than the others. In addition, comparison of the inhibition rate of 3-methyl-canthin-2,6-one (61) and 3-methoxy-canthin-2,6-one (64) indicated that the methoxy group at N-3 may improve the inhibitory activity.
In summary, an alkaloidal compound library from Simaroubaceae and their chemically synthesized analogues were assayed for their potential anti-inflammatory activity. The results demonstrated that a number of β-carboline-type and canthinone-type alkaloids from the compound library exhibited potent activity suppressing the overproduction of NO in LPS-activated RAW 264.7 macrophages. Based on further investigation on the selected six bioactive alkaloids, we found that two canthinone-type alkaloids suppressed the release of NO and PGE2 through down-regulating the expression of inflammatory proteins iNOS and COX-2. Four β-carboline-type alkaloids suppressed the production of NO via down-regulation of iNOS expression, but have no effect on the release of PGE2 and are independent of COX-2 pathway. Both the β-carboline-type alkaloids and the canthinone-type alkaloids have been identified as anti-inflammatory agents of Simaroubaceae plants, but these two types of natural compounds showed entirely different inhibitory profiles on the expression of iNOS and COX-2 in LPS-activated RAW 264.7 macrophages. Further detailed investigations on their modulating effect on the cellular signal transduction pathways such as NF-κB or MAPK pathways are expected to certify the differences between the mechanism of β-carboline-type alkaloids and canthinone-type alkaloids.
Materials and methods
Macrophage RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Roswell Park Memorial Institute (RPMI) 1640 medium was purchased from GE Healthcare Life Sciences. Fetal bovine serum (FBS) was purchased from NQBB International Biological Corporation. LPS (from E. coli), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and DMSO were purchased from Sigma–Aldrich, Inc. (St. Louis, MO, USA). HSS is a product of Tianjin Biochem Pharmaceutical Co., Ltd. (Tianjin, China). The mouse PGE2 ELISA kit was purchased from Shanghai Senxiong Science and Technology Industry Co., Ltd. (Shanghai, China). Mouse anti-rabbit inducible nitric oxide synthase (iNOS) polyclonal antibody (catalog no. 160862) and mouse anti-rabbit COX-2 polyclonal antibody (catalog no. 160106) were purchased from Cayman Chemical Company. Goat anti-rabbit β-actin polyclonal antibody (catalog no. sc-1616) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
All test compounds were dissolved in cell culture-grade DMSO at the concentration of 50 mM as a stock solution and stored at −20 °C. The stock solution was diluted to indicate concentrations by DMSO before use. The final concentration of DMSO in the medium was 0.2%. HSS, a clinical anti-inflammatory drug, was used as the positive control. HSS was dissolved in RPMI 1640 medium at the concentration of 50 mM as a stock solution and stored at − 20 °C. The stock solution was diluted to indicate concentrations by RPMI 1640 medium before use.
Cell culture of RAW 264.7
Mouse monocyte-macrophage RAW 264.7 cells (ATCC TIB-71) were fostered in RPMI 1640 medium mixed with 10% heat-inactivated FBS at 37 °C in an incubator with 5% CO2. The medium was routinely replaced every day and RAW 264.7 cells were passaged until they reached about 80% of confluence.
Nitric oxide analysis and cell viability assay
The cells were prepared at a density of 1 × 106 cells/ml, and 200 μl was seeded in each well of the 96-well plates. The cells were treated by LPS (1 μg/ml; Sigma–Aldrich, St. Louis, MO, USA) with or without indicated concentrations of test compounds for 24 h. Cell culture supernatant (100 μl) was then removed to another 96 well-plate and mixed with 100 μl of Griess reagent containing equal volumes of Griess reagent A: 0.1% (w/v) of N-(1-naphthyl) ethylenediamine solution and Griess reagent B: 1% (w/v) sulfanilamide in 5% (v/v) H3PO4 solution (Yantai Science and Biotechnology Co., Ltd., Yantai, China). After being mixed for 10 min, the absorbance was measured at 540 nm using a microplate reader. The nitrite concentrations were calculated according to the method reported by Jin et al. .
After 100 μl of the cell culture supernatant was taken out for NO determination, MTT solution (5 mg/ml) was added in the original 96-well plate at the final concentration of 200 μg/ml and then incubated for 4 h. After removal of the supernatant from the 96-well plate, 150 μl of DMSO was added to dissolve the formazan. The absorbance was measured by a microplate reader at a wavelength of 570 nm, and a wavelength of 655 nm was used as reference. The untreated cells were considered to be 100% viable. Final results are expressed as percentage of viable cells in the experimental group when compared with those of the untreated group.
Determination of PGE2
RAW 264.7 cells were prepared at a density of 1 × 106 cells/ml, and 200 μl was seeded in each well of the 96-well plates. The cells were treated by LPS (1 μg/ml) with or without indicated concentrations of test compounds for 24 h. Cell culture supernatant (100 μl) was removed to measure the level of PGE2 by using a commercial mouse PGE2 ELISA kit (Shanghai Senxiong Science and Technology Industry Co., Ltd., Shanghai, China) according to the manufacturer’s instructions.
Western blot analysis of iNOS, COX-2 and β-actin proteins
The cells were seeded in 60-mm cell culture dishes for 1 h, and then treated by LPS (1 μg/ml) with or without indicated compounds (12.5, 25, and 50 μM) for 24 h. The cells were washed with cold PBS and lysed in a cold lysis buffer, and the total protein was extracted from ultrasonic crushed cells. The total protein and the cellular debris were separated by centrifugation at 13,000 rpm for 6 min. The protein concentrations were determined by a commercial Bradford protein assay kit. Total protein (30 μg) was separated by 8% polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. The membranes were blocked with Tris-buffered saline with 0.5% triton X-100 (TBS-T) containing 5% skim milk at room temperature for 4 h. The membranes were then washed with TBS-T for three times and incubated overnight at 4 °C with anti-iNOS, anti-COX-2, or anti-β-actin solution which was diluted with TBS-T. After washing with TBS-T, the membranes were incubated for 1 h at room temperature with horseradish peroxidase (HRP)-labeled goat anti-murine IgG (H+L), or HRP-labeled goat anti-rabbit IgG (H+L) as secondary antibody diluted with TBS-T, respectively. The bands were detected with an enhanced chemiluminescence system and the bands representing iNOS, COX-2, and β-actin were quantitated by DigDoc100 program (Gel pro analyzer 3.2). The levels of corresponding iNOS and COX-2 were normalized on the basis of the corresponding β-actin levels.
The data are expressed as mean ± SD. The results were assessed by one-way analysis of variance with the SPSS 16.0 statistical program. p ≤ 0.05 was considered to indicate a statistically significant difference.
The research of the present study was supported by the National Natural Science Foundation of China Programs (grant nos. 81102781, 81274039, and 81573614), the Shandong Provincial Natural Science Foundation (grant no. ZR2016HL54), the National college students’innovation and entrepreneurship training program (201811066014), and the Undergraduate Scientific and Technological Innovation Project of Yantai University (201811066025).
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
- 11.Fynn PM, Opoku-Boahen Y, Adukpo GE, Armah FA (2016) Anti-inflammatory and antioxidant activities of canthinone alkaloids from Anthostema aubryanum (Baill). Nat Prod Plant Resour 6:13–24Google Scholar
- 14.Jiao WH, Gao H, Li CY, Zhou GX, Kitanaka S, Ohmurae A, Yao XS (2010) β-Carboline alkaloids from the stems of Picrasma quassioides. Magn Reson Chem 48:490–495Google Scholar
- 15.Zhu CC, Deng GH, Lin CZ (2012) Chemical constituents of Picrasma quassioides (D. Don) Benn. Nat Prod Res Dev 24:476–478Google Scholar
- 16.Zhao W, Chen ZW, Sun JH, He Y, Liu W, Hu S, Guan ZY, Chen FL (2012) Alkaloid extraction from Picramia quassioides (D. Don) Benn. and safety of its compound injection. J South Agric 43:865–868Google Scholar
This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.