Bioactivity-Guided isolation of β-Sitosterol and Some Fatty Acids as Active Compounds in the Anxiolytic and Sedative Effects of Tilia americana var. mexicana

Abstract

Tilia species have been used as anxiolytics for many years. In a previous study anxiolytic-like effects of a hexane extract of Tilia americana var. mexicana inflorescences were observed in experimental models in mice. To get additional insights into the neuroactive actions of this particular Tilia species, in this study we report a bioactivity guided-fractionation of the extract and separation by column chromatographic methods to isolate three fatty acids and a triterpene identified as β-sitosterol as major constituents. Our results revealed that the crude extract at 10 and 30 mg/kg i. p. and some pooled fractions at the same dosages potentiated sodium pentobarbital-induced sleeping time and caused a significant increase in the time spent at the open-arm sides in the plus-maze test. A reduction in the exploratory behavioral pattern manifested as ambulatory activity, as well as head dipping and rearing tests was also observed. Further fractionation and purification yielded four major fractions containing fatty acids and β-sitosterol as the active compounds. A dose-response curve of β-sitosterol in the range 1 to 30 mg/kg doses indicated that this compound produced an anxiolytic-like action from 1 to 10 mg/kg and a sedative response when the dose was increased to 30 mg/kg, these effects resemble those produced by diazepam (0.1 mg/kg). Our results suggest that hexane extract of Tilia americana var. mexicana produces depressant actions on the central nervous system, at least in part, because of the presence of β-sitosterol and some fatty acids that remain to be identified.

Key words

Anxiety - β-sitosterol - central nervous system - sedative - Tilia americana var. mexicana - Tiliaceae

References

• 1 Miller L, Murray W. Herbal medicinal: a clinician’s guide. New York; Pharmaceutical Products Press 1998.
• 2 McCann J. Herbal medicine handbook. 2nd edition Philadelphia; Lippincott 2003.
• 3 Argueta A, Cano L, Rodarte M. Atlas de las plantas de la medicina tradicional mexicana. Mexico; Instituto Nacional Indigenista 1994: 1193.
• 4 Coleta M, Campos M G, Cotrim M D, Cunha A P. Comparative evaluation of Melissa officinalis L., Tilia europea L., Passiflora edulis Sims. and Hypericum perforatum L. in the elevated plus maze anxiety test.  Pharmacopsychiatry. 2001;  34 520-1.
• 5 Viola H, Wolfman C, Levi de Stein M, Wasowski C, Peña C, Medina J H. et al . Isolation of pharmacologically active benzodiazepine receptor ligands from Tilia tomentosa (Tiliaceae).  J Ethnopharmacol. 1994;  44 47-53.
• 6 Buchbauer G, Remberg B, Jirovetz L, Nikiforov A. Comparative headspace analysis of living and fresh cut lime tree flowers (flores Tiliae).  Flavour Fragance J. 1995;  10 221-4.
• 7 Toker G, Aslan M, Yesilada E, Memisoglu M, Ito S. Comparative evaluation of the flavonoid content in officinal Tiliae flos and Turkish lime species for quality assessment.  J Pharm Biomed Anal. 2001;  26 111-21.
• 8 Kram G, Franz G. Structural investigations on water-soluble polysaccharides of lime three flowers (Tilia cordata L.)  Pharmazie. 1985;  40 501-2.
• 9 Medina J H, Viola H, Wolfman C, Marder M, Wasowski C, Calvo D. et al . Overview - flavonoids: a new family of benzodiazepine receptor ligands.  Neurochem Res. 1997;  22 419-25.
• 10 Aguirre-Hernández E, Martínez A L, González-Trujano M E, Moreno J, Vibrans H, Soto-Hernández M. Pharmacological evaluation of the anxiolytic and sedative effects of Tilia americana L. var. mexicana in mice.  J Ethnopharmacol. 2007;  109 140-5.
• 11 González-Trujano M E, Martínez A L, Reyes-Ramirez A, Reyes-Trejo B, Navarrete A. Palmitone isolated from Annona diversifolia induces an anxiolytic-like effect in mice.  Planta Med. 2006;  72 703-7.
• 12 González-Trujano M E, Navarrete C A, Reyes B, Hong E. Some pharmacological effects of the ethanol extract of leaves of Annona diversifolia on the central nervous system in mice.  Phytother Res. 1998;  2 600-2.
• 13 Ha J H, Lee M G, Chang S M, Lee J T. In vivo characterization of sedative activities of Fossilia mastodi Ossis.  Biol Pharm Bull. 2006;  29 1414-7.
• 14 Lister R G. The use of a plus-maze to measure anxiety in the mouse.  Psychopharmacology. 1987;  92 180-5.
• 15 Clark G, Koster A G, Person D W. Exploratory behaviour in chronic disulfotan poisoning in mice.  Psychopharmacologia (Berl). 1997;  20 169-71.
• 16 Ugalde M, Reza V, Gonzalez-Trujano M E, Avula B, Khan I A, Navarrete A. Isobolographic analysis of the sedative interaction between six central nervous system depressant drugs and Valeriana edulis hydroalcoholic extract in mice.  J Pharm Pharmacol. 2005;  57 631-40.
• 17 Pelletier X, Belbraouet S, Mirabel D, Mordret F, Perrin J L, Pages X. et al . A diet moderately enriched in phytosterols lowers plasma cholesterol concentrations in normocholesterolemic humans.  Ann Nutr Metab. 1995;  39 291-5.
• 18 Villaseñor I M, Angelada J, Canlas A P, Echegoyen D. Bioactivity studies on beta-sitosterol and its glucoside.  Phytother Res. 2002;  16 417-21.
• 19 Berges R R, Windeler J, Trampisch H J, Senge T. Randomised, placebo-controlled, double-blind clinical trial of beta-sitosterol in patients with benign prostatic hyperplasia. Beta-sitosterol study group.  Lancet. 1995;  345 1529-32.
• 20 Chattopadhyay D, Arunachalam G, Mandal S C, Bhadra R, Mandal A BJ. CNS activity of the methanol extract of Mallotus peltatus (Geist) Muell Arg. leaf: an ethnomedicine of Onge.  J Ethnopharmacol. 2003;  85 99-105.
• 21 López-Rubalcava C, Pina-Medina B, Estrada-Reyes R, Heinze G, Martinez-Vazquez M. Anxiolytic-like actions of the hexane extract from leaves of Annona cherimolia in two anxiety paradigms: possible involvement of the GABA/benzodiazepine receptor complex.  Life Sci. 2006;  78 730-7.
• 22 Gyamfi M A, Hokama N, Oppong-Boachie K, Aniya Y. Inhibitory effects of the medicinal herb, Thonningia sanguinea, on liver drug metabolizing enzymes of rats.  Hum Exp Toxicol. 2000;  19 623-31.
• 23 Sivam S P, Nabeshima T, Ho I K. Acute and chronic effects of pentobarbital in relation to postsynaptic GABA receptors: A study with muscimol.  J Neurosci Res. 1982;  7 37-47.
• 24 Mikolajczyk T, Roelants I, Epler P, Ollevier F, Schulz R, Sokolowska-Mikolajczyk M. et al . Assessment of tissue damaging effects of mixed micellar absorption enhancers on the intestinal mucosa of common carp (Cyprinus carpio), African catfish (Clarias gariepinus) and rainbow trout (Oncorhynchus mykiss) as a consequence of enhanced intestinal absorption of sGnRH-a.  J Appl Ichthyol. 2001;  17 267-72.
• 25 Donegan L, Hudye S A, Olson M E, Hardin J A, Gall D G. Effect of a dietary nonionic surfactant on small intestinal nutrient transport.  Dig Dis Sci. 1999;  44 1423-7.
• 26 Dong-Qin Q, Gui-Xia X, Xiang-Gen W. Formulation development and pharmacokinetics of puerarin self-emulsifying drug delivery systems.  PDA J Pharm Sci Technol. 2007;  61 37-43.
• 27 Haefely W E, Facklam M, Schoch P, Martin J R, Bonetti E P, Moreau J L. et al . Partial agonists of benzodiazepine receptors for the treatment of epilepsy, sleep, and anxiety disorders.  Adv Biochem Psychopharmacol. 1992;  47 379-94.
• 28 Tallman J F, Thomas J W, Gallager D W. GABAergic modulation of benzodiazepine binding site sensitivity.  Nature. 1978;  274 383-5.
• 29 File S E. The contribution of behavioral studies to the neuropharmacology of anxiety.  Neuropharmacology. 1987;  26 877-86.
• 30 Lydiard R B. The role of GABA in anxiety disorders.  J Clin Psychiatry. 2003;  64 21-7.