Cytotoxicity, nitric oxide and acetylcholinesterase inhibitory activity of three limonoids isolated from Trichilia welwitschii (Meliaceae)
© Dzoyem et al. 2015
Received: 13 July 2015
Accepted: 1 October 2015
Published: 13 October 2015
Limonoids are highly oxygenated compounds with a prototypical structure. Their occurrence in the plant kingdom is mainly confined to plant families of Meliaceae and Rutaceae. Owing to their wide range of pharmacological and therapeutic properties, this study was aimed at investigating the potential nitric oxide (NO) and acetylcholinesterase (AChE) inhibitory activity and the cytotoxicity of three limonoids: trichilia lactone D5 (1), rohituka 3 (2) and dregeanin DM4 (3), isolated from Trichilia welwitschii C.DC.
Results indicated that the three limonoids had low cytotoxicity towards Vero cells with LC50 values ranging from 89.17 to 75.82 μg/mL. Compounds (2) and (3) had lower cytotoxicity compared to puromycin and doxorubicin used as reference cytotoxic compounds. Compound (1) (LC50 of 23.55 μg/mL) had good antiproliferative activity against RAW 264.7 cancer cells. At the lowest concentration tested (0.5 µg/mL), compound (2) and (3) released the lowest amount of nitric oxide (2.97 and 2.93 µM, respectively). The three limonoids had anti-AChE activity with IC50 values ranged of 19.13 μg/mL for (1), 34.15 μg/mL for (2) and 45.66 μg/mL for (3), compared to galantamine (IC50 of 8.22 μg/mL) used as positive control.
The limonoid compounds studied in this work inhibited nitric oxide production in LPS-stimulated macrophages and had anti-AChE activity. Trichilia lactone D5 had potential antiproliferative activity against RAW 264.7 cancer cells. The limonoids had low cytotoxicity towards Vero cells lines. This study provided further examples of the importance of limonoids compounds as potential AChE inhibitors and anti-inflammatory agents targeting the inhibition of NO production.
KeywordsTrichilia welwitschii Cytotoxicity Acetylcholinesterase Nitric oxide Limonoids
Nitric oxide (NO) is an important pro-inflammatory mediator involved in a wide variety of physiological and pathophysiological events; however overproduction of NO by inducible nitric oxide synthase (iNOS) results in severe inflammation . An association between the development of cancer and inflammation has long-been documented . Moreover, inflammation accelerates the appearance of some neurodegenerative disorders, such as Parkinson and Alzheimer’s diseases . These insights are fostering new anti-inflammatory therapeutic approaches to cancer and neurodegenerative diseases development. There is no doubt that natural products remain important sources of new pharmaceutical compounds. Therefore, natural products research continues to explore a variety of compounds which may be used for the development of new drugs.
Limonoids are highly oxygenated compound with a prototypical structure, either containing or derived from a precursor with a 4,4,8-trimethyl-17-furanylsteroid skeleton. The prototypical structure consists of four six-membered rings and a furan ring. It is an important group of metabolically altered triterpenes, which are limited in their distribution. Their occurrence in the plant kingdom is mainly confined to plant families of Meliaceae and Rutaceae, and occurs less frequently in Cneoraceae and Harrisonia sp. of Simaroubaceae . Limonoids isolated from the plant family Meliaceae are more complex with very high degree of oxidation and structural rearrangements . In recent years a large number of pharmacological studies have been carried out to indicate their beneficial effects. Medicinal properties of limonoids reported include antibacterial, antifungal, antimalarial, anticancer and antiviral activities [6, 7]. In the last years, more than 100 limonoids have been isolated and characterized . In our previous study, we reported the isolation and characterization of three limonoids compounds (dregeanin DM4, rohituka 3 and trichilia lactone D5) from Trichilia welwitschii C.DC. .
Trichilia welwitschii is a West African member of the Meliaceae growing as a large tree in the Terra Firma Forests of Nigeria, Cameroon, Angola and Gabon . Species from the Meliaceae family and especially Trichilia genus have been well-documented for their ability to metabolize structurally diverse and biologically significant triterpenoids and limonoids . No previous pharmacological study has been reported on compounds isolated from T. welwitschii. In our continue search of bioactive compounds from plants and owing to the wide range of pharmacological and therapeutic properties of limonoids, this study was carried out to investigate the potential antiproliferative, nitric oxide and acetylcholinesterase inhibitory activity of three limonoids isolated from T. welwitschii.
Results and discussion
Cytotoxicity (LC50 in µg/mL) and the selectivity index (SI) of three limonoids isolated from Trichilia welwitschii and reference compounds (doxorubicin and puromycin) against cancer cell lines
89.17 ± 5.00a
81.20 ± 6.38a
23.55 ± 5.77a
85.22 ± 6.31a
81.20 ± 4.04a
65.68 ± 3.64b
75.82 ± 1.85b
84.53 ± 5.81a
61.86 ± 4.14b
9.35 ± 0.66c
1.06 ± 0.65c
5.32 ± 0.90d
0.4 ± 0.02b
1.15 ± 0.17c
NO inhibitory activity
Acetylcholinesterase inhibitory activity of three limonoids isolated from Trichilia welwitschii and reference compound (galantamine)
% AChE inhibition
94.33 ± 8.15
19.13 ± 0.41a
71.67 ± 6.03
18.00 ± 8.89
28.00 ± 6.56
82.67 ± 7.04
34.15 ± 1.66b
67.00 ± 4.36
47.33 ± 2.08
14.00 ± 6.00
87.00 ± 8.74
45.69 ± 3.65c
70.67 ± 3.50
31.67 ± 5.20
30.33 ± 3.51
83.00 ± 8.89
8.22 ± 2.73d
69.67 ± 4.16
57.67 ± 4.04
38.00 ± 1.00
The limonoid compounds studied in this work inhibited nitric oxide production in LPS-stimulated macrophages and presented AChE inhibitory activity. They had low cytotoxicity against Vero cells lines. The potential antiproliferative effect of compound against RAW 264.7 cancer cells was also demonstrated. This study provided further examples of the importance of limonoid compounds as potential AChE inhibitors and anti-inflammatory agents targeting the inhibition of NO production.
Sodium dodecyl sulphate, bovine serum albumin (BSA), sodium chloride (NaCl), MgCl2·6H2O, acetylthiocholine iodide (ATCI), galantamine, 5,5-dithiobis-2-nitrobenzoic acid (DTNB), acetylcholinesterase (AChE) enzyme from electric eels (type VI-S lyophilized powder), sodium nitrite, ferrous sulfate, indomethacin and 15-lipoxygenase from Glycine max purchased from Sigma (Germany) and Tris(hydroxymethyl)aminomethane from Sigma, (Switzerland). Foetal calf serum (FCS), penicillin/streptomycin/fungizone (PSF) and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Highveld Biological Products (South Africa). Phosphate buffered saline (PBS) and trypsin were purchased from Whitehead Scientific (South Africa). Quercetin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich St. Louis, MO, USA.
Cells lines including human monocytic THP-1, murine macrophage RAW 264.7 and the Vero monkey kidney cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA). They were maintained in DMEM supplemented with 10 % fetal calf serum (FCS) and 1 % penicillin/streptomycin/fongizone (PSF) under standard cell culture conditions at 37 °C and 5 % CO2 in a humidified environment.
The cytotoxicity of compounds was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) assay as previously described . The selectivity index (SI) values were calculated by dividing cytotoxicity LC50 values of normal Vero cells by the LC50 of cancer cells in the same units.
Nitric oxide inhibitory activity and viability of LPS-activated RAW 264.7 macrophages
The RAW 264.7 macrophages cells were seeded in 96 well-microtitre plates and were activated by incubation in medium containing 1 µg/mL LPS alone (control) or lipopolysaccharide with different concentrations of the samples dissolved in DMSO. Quercetin served as a positive control NO inhibitor for the reduction of NO production .
Measurement of nitrite
Nitric oxide released from macrophages was determined by measuring the nitrite concentration in culture supernatant using the Griess reagent. After 24 h incubation, 100 µL of supernatant from each well of cell culture plates was transferred into 96-well microtitre plates and an equal volume of Griess reagent was added. The absorbance of the resultant solutions was determined on a BioTek Synergy microplate reader after 10 min at 550 nm. The concentrations of nitrite were derived from regression analysis using serial dilutions of sodium nitrite as a standard. Percentage inhibition was calculated based on the ability of compounds to inhibit nitric oxide formation by cells compared with the control (cells in media without compounds), which was considered as 0 % inhibition.
To determine whether the observed nitric oxide inhibition was not due to cytotoxic effects, MTT assay was also performed on the macrophage cells as previously described .
Acetylcholinesterase inhibition activity
All results are presented as means of triplicate experiments. All experiments were conducted in triplicate and values expressed as mean ± standard deviation. Statistical analysis was performed with GraphPad InStat Software and results were compared using the Student-Newman Keul test at 5, 1 or 0.1 % significance level.
Dulbecco’s modified Eagle’s medium
JPD performed experiments and wrote the first draft of manuscript. ATT and RM isolated the compounds. PM and AENkengfack and supervised the chemistry part of the work. JNE and LJM supervised the biological part of work and revised the final manuscript. All authors read and approved the final manuscript.
The University of Pretoria provided a postdoctoral fellowship to JPD. The National Research Foundation (NRF) and Medical Research Council (MRC) provided funding to support this study.
The authors declared that they have no competing interests.
Open AccessThis 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. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Hirai S, Horii S, Matsuzaki Y, Ono S, Shimmura Y, Sato K, Egashira Y. Anti-inflammatory effect of pyroglutamyl-leucine on lipopolysaccharide-stimulated RAW 264.7 macrophages. Life Sci. 2014;117(1):1–6.View ArticlePubMedGoogle Scholar
- Coussens LM, Werb Z. Inflammation and cancer. Nature. 2012;2012(420):860–7.Google Scholar
- De Pablos RM, Espinosa-Oliva AM, Sarmiento M, Venero JL. Stress and inflammation: a detrimental combination in the development of neurodegenerative disease. Inflamm Cell Signal. 2014;1:e182.Google Scholar
- Roy A, Saraf S. Limonoids: overview of significant bioactive triterpenes distributed in plants kingdom. Biol Pharm Bull. 2006;29:191–201.View ArticlePubMedGoogle Scholar
- Pekala J, Strub DJ, Koziol A, Lochynski S. Variability of Biological Activities of Limonoids Derived from Plant Sources. Mini-Rev Org Chem. 2015;11:269–79.Google Scholar
- Miller EG, Porter JL, Binnie WH, Guo IY, Hasegawa S. Further studies on the anticancer activity of citrus limonoids. J Agric Food Chem. 2004;52:4908–12.View ArticlePubMedGoogle Scholar
- Champagne DE, Koul O, Isman MB, Scudder GGE, Towers GHN. Biological activity of limonoids from the rutales. Phytochemistry. 1992;31:377–94.View ArticleGoogle Scholar
- Tundis R, Loizzo MR, Menichini F. An overview on chemical aspects and potential health benefits of limonoids and their derivatives. Crit Rev Food Sci Nutr. 2014;54:225–50.View ArticlePubMedGoogle Scholar
- Tsamo A, Langat MK, Nkounga P, Waffo AFK, Nkengfack AE, Mulhollan DA. Limonoids from the West African Trichilia welwitschii (Meliaceae). Biochem Syst Ecol. 2013;50:368–70.View ArticleGoogle Scholar
- Louppe D, Oteng-Amoaka AA, Brink M. (Eds.), Plant resources of tropical Africa. Timbers 1. PROTA Foundation. Leiden: Blackhuys Publishers, 2008;7(1):562–3.
- Curcino V, da Silva TW, dos Santos GM, Braz-Filho R. Secondary metabolites of the genus Trichilia: contribution to the chemistry of Meliaceae family. Am J Anal Chem. 2014;5:91–121.View ArticleGoogle Scholar
- Söderlund G, Haarhaus M, Chisalita S, Arnqvist HJ. Inhibition of puromycin-induced apoptosis in breast cancer cells by IGF-I occurs simultaneously with increased protein synthesis. Neoplasma. 2004;51(1):1–11.PubMedGoogle Scholar
- Tacar O, Sriamornsak P, Dass CR. Doxorubicin: an update on anticancer molecular action, toxicity and novel drug delivery systems. J Pharm Pharmacol. 2013;65(2):157–70.View ArticlePubMedGoogle Scholar
- Pan X, Matsumoto M, Nishimoto Y, Ogihara E, Zhang J, Ukiya M, Tokuda H, Koike K, Akihisa M, Akihisa T. Cytotoxic and nitric oxide production-inhibitory activities of limonoids and other compounds from the leaves and bark of Melia azedarach. Chem Biodivers. 2014;11(8):1121–39.View ArticlePubMedGoogle Scholar
- Takagi M, Tachi Y, Zhang J, Shinozaki T, Ishii K, Kikuchi T, Ukiya M, Banno N, Tokuda H, Akihisa T. Cytotoxic and melanogenesis-inhibitory activities of limonoids from the leaves of Azadirachta indica (Neem). Chem Biodivers. 2014;11(3):451–68.View ArticlePubMedGoogle Scholar
- Kikuchi T, Ishii K, Noto T, Takahashi A, Tabata K, Suzuki T, Akihisa T. Cytotoxic and apoptosis-inducing activities of limonoids from the seeds of Azadirachta indica (neem). J Nat Prod. 2011;74(4):866–70.View ArticlePubMedGoogle Scholar
- Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 1991;43(2):109–42.PubMedGoogle Scholar
- Mu MM, Chakravortty D, Sugiyama T, Koide N, Takahashi K, Mori I, Yoshida T, Yokochi T. The inhibitory action of quercetin on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells. J Endotoxin Res. 2001;7(6):431–8.View ArticlePubMedGoogle Scholar
- Sarigaputi C, Sommit D, Teerawatananond T, Pudhom K. Weakly anti-inflammatory limonoids from the seeds of Xylocarpus rumphii. J Nat Prod. 2014;77(9):2037–43.View ArticlePubMedGoogle Scholar
- Samochocki M, Höffle A, Fehrenbacher A, Jostock R, Ludwig J, Christner C, Radina M, Zerlin M, Ullmer C, Pereira EF, Lübbert H, Albuquerque EX, Maelicke A. Galantamine is an allosterically potentiating ligand of neuronal nicotinic but not of muscarinic acetylcholine receptors. J Pharmacol Exp Ther. 2003;305(3):1024–36.View ArticlePubMedGoogle Scholar
- Amoo SO, Aremu AO, Moyo M, Van Staden J. Antioxidant and acetylcholinesterase-inhibitory properties of long-term stored medicinal plants. BMC Complement Altern Med. 2012;12:87.PubMed CentralView ArticlePubMedGoogle Scholar
- Jabeen B, Riaz N, Saleem M, Naveed MA, Ahmed M, Tahir MN, Pescitelli G, Ashraf M, Ejaz SA, Ahmed I, Jabbar A. Isolation and characterization of limonoids from Kigelia Africana. Z Naturforsch. 2013;68b:1041–8.Google Scholar
- Dzoyem JP, McGaw LJ, Eloff JN. In vitro antibacterial, antioxidant and cytotoxic activity of acetone leaf extracts of nine under-investigated Fabaceae tree species leads to potentially useful extracts in animal health and productivity. BMC Complement Altern Med. 2014;14:147.PubMed CentralView ArticlePubMedGoogle Scholar
- Ellman GL, Courtney KD, Andres V Jr, Featherstone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961;7:88–95.View ArticlePubMedGoogle Scholar