Phytochemical Study and Evaluation of Antioxidant, Neuroprotective and Acetylcholinesterase Inhibitor Activities of Galeopsis ladanum L. extracts
Pueyo I Uriarte, MI Calvo
Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Navarra, 31008 Pamplona (Navarra), Spain
|Date of Web Publication||8-Dec-2009|
Pueyo I Uriarte
Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Navarra, 31008 Pamplona (Navarra)
Source of Support: None, Conflict of Interest: None
| Abstract|| |
The antioxidant activity, neuroprotective effect and acetylcholinesterase activity of the dichloromethane, ethyl acetate, methanol and water extracts of seeds, leaves and roots of Galeopsis ladanum L. were investigated. Ethyl acetate, methanol and water extracts of leaves showed the highest antioxidant activity (DPPH). Methanol and water extracts of seeds and the water extract of roots showed neuroprotective effect on hydrogen peroxide induced apoptosis in rat pheochromocytoma PC12 cells. Some dichloromethane, ethyl acetate and water extracts exhibited antiacetylcholinesterase activity by TLC.
Keywords: Galeopsis ladanum ; antioxidant activity ; neuroprotective effect ; PC12 cells ; acetylcholinesterase inhibitors
|How to cite this article:|
Uriarte PI, Calvo M I. Phytochemical Study and Evaluation of Antioxidant, Neuroprotective and Acetylcholinesterase Inhibitor Activities of Galeopsis ladanum L. extracts. Phcog Mag 2009;5:287-90
|How to cite this URL:|
Uriarte PI, Calvo M I. Phytochemical Study and Evaluation of Antioxidant, Neuroprotective and Acetylcholinesterase Inhibitor Activities of Galeopsis ladanum L. extracts. Phcog Mag [serial online] 2009 [cited 2021 Oct 21];5:287-90. Available from: http://www.phcog.com/text.asp?2009/5/20/287/58146
| Introduction|| |
In recent years considerable attention has been devoted to medicinal plants with antioxidant properties reducing free radical produced tissue injury. In living organisms the reactive oxygen species (ROS) and reactive nitrogen species (RNS) are known to cause damage to lipids, proteins, enzymes, and nucleic acids leading to cell or tissue injury implicated in the processes of aging as well as in wide range of degenerative diseases including inflammation, cancer, atherosclerosis, diabetes, liver injury, Alzheimer, Parkinson, and coronary heart pathologies, among others  .
In the present study, we examined the protective effects of G. ladanum extracts on H 2 O 2 induced cytotoxicity in cultured rat pheochromocytoma PC12 cells that retain dopaminergic characteristics and have been widely used for neuroprotective studies ,, .
Alzheimer's disease (AD) is the most common cause of senile dementia in elderly humans. Actually acetylcholinesterase inhibitors (AChEI) are the most important compounds for the treatment of the AD symptoms, increasing the neurotransmitter acetylcholine levels at cerebral cortex synapses. Current efforts to identify new AChEI are mostly focused on alkaloids, but day after day are more studies of non-alkaloids compounds. There have been described flavonoids, xanthones, chalcones, cumarins and terpenoids with this activity ,, .
Galeopsis ladanum L. was previously examined for chemical constituents ,, . In these studies were identified 16 apigenin, luteolin, scutellarein, isoscutellarein and hipolaetin derived flavonoids, caffeic acid, pseudochlorogenic acid, stachydrine, harpagoside and 8-O-acethylharpagoside. Flavonoids as linariin and isolariin that showed AChEI activity ,, , have similar chemical structure to flavonoids presents in G. ladanum .
| Materials and Methods|| |
Preparation of the Extracts
Seeds, leaves and roots of Galeopsis ladanum L. ( Lamiaceae ) were collected in Congosto de Valdavia, Palencia (Spain). The plant was identifi ed by Dr. R.Y. Cavero, Department of Plant Biology of the University of Navarra, Spain. Dry powdered plant material (seeds, leaves and roots) was extracted by sequential cold maceration using dichloromethane, ethyl acetate, methanol and water.
Preliminary Phytochemical Analysis
All extracts were subjected to preliminary phytochemical screening for the determination of major chemical groups by TLC  and HPLC. Determination of Total Phenols was performance employing Prussian Blue method  , recommended by European Pharmacopoeia.
Free Radical Scavenging Activity
Antioxidant activity of plant extracts was determined through spectrophotometry using 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging radical assay  . Extracts were prepared in their correspondent solvent to obtain concentrations of 10, 20, 50, 100, 200, 400, 800, 1200 and 1600 μg/ml; and standards at concentrations of 0.25, 0.5, 1, 2, 5, 10, 20, 40 and 100 μg/ml. 150 μl of a 0.04 mg/ml MeOH solution of DPPH were added to 150 μl of diluted solutions. The absorbance was determined at different times (0, 15, 30, 45, 60 and 75 min) at 517 nm. % inhibitions were plotted against respective concentration to obtain correspondent IC 50 . These results were plotted against respective time to obtain IC 50max (IC 50 of maximum inhibition) and t max (time of maximum inhibition). The experiment was performed in quadruplicate. Vitamin C and BHA were used as positive controls.
Cytotoxicity and Neuroprotective Effect
Cytotoxicity and neuroprotective effect were performed in rat pheochromocytoma PC12 cells. For the determination of the cytotoxicity, cells were seeded in 96 multiwell plates at a density of 20.000 cells/well and incubated during 48 hours. 100 μl of different extract solutions (1, 10, 50, 100, 250, 500 y 1000 μg/ml in DEMEM supplemented with horse serum 1 %) were added to the wells and incubated 24 hours. 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) is an indicator of the mitochondrial activity of living cells. MTT is reduced to a coloured compound (formazan) by mitochondria. The free-cell medium was removed and 100 μl of MTT-PBS horse serum 1 % solution (0.4 mg/ml fi nal concentration) was added. After 1 h of 37 0 Cincubation, MTT solution was removed and formazan was dissolved in 100 μl of DMSO . MTT reduction in living cells was quantified at 570 nm wavelength using a Power Wave XS microplate reader. The experiment was performed in quadruplicate. For the determination of the neuroprotective activity, cells were treated for 30 min with 100 μl of PBS containing H2 O 2 (0.2 mM) 24 hours after the addition of the extracts employing the same conditions that in the cytotoxicity test and using the MTT assay for the determination of cell survival  .
Acetylcholinesterase Inhibitor Activity
Antiacetylcholinesterase activity was studied by TLC with galanthamine as reference compound using Ellman's method  . TLC plates were sprayed DTNB or 5,5′- dithiobis(2-nitrobenzoic acid)/ATCI or acetylthiocholine iodide 1:1 (1 mM DTNB and 1 mM ATCI in tris-HCl 50 mM buffer pH 8). It was allowed to dry for 3-5 min and then 3 U/ml of acetylcholinesterase (AchE) solution was sprayed. In order to verify whether the positive results shown using Ellman′s method were due to enzyme inhibition or to chemical inhibition, we employed the false positive method developed by Rhee et al  . Firstly TLC plates were sprayed with a 1 mM solution of DTNB followed by the thiocholine spray. Tiocholine was obtained incubating for 15 min at 37°C a mixture of 3 U/ml of AchE and 1 mM ATCI in tris-HCl 50 mM buffer (pH 8). Galanthamine was used as standard and the results were compared with the results of Ellman′s method.
| Results and Discussion|| |
The phytochemical analysis by TLC and HPLC showed the presence of flavonoids, phenolic acids, alkaloids, iridoids and saponins in different extracts [Table 1]. Fatty acids are the major compounds in seeds. Alkaloids were only present in methanolic extracts of all plant organs, while saponins only in aqueous extracts. Flavonoids were detected principally in ethyl acetate and methanolic extracts of leaves and roots, and phenolic acids in ethyl acetate, methanolic and aqueous extracts of all plant organs.
Flavonoids were and identified by HPLC-UV as isoscutellarein derivates (5, 7, 8, 4′-tetrahydroxyflavone), λ MeOH = .277, 305, 326 sh, and hypolaetin derivatives (5, 7, 8, 3′,4′- pentahydroxyflavone), (λ MeOH = 277, 300 sh, 340). Ethyl acetate (16.5 μg/ml), methanol (21.3 μg/ml) and water (37.1 μg/ml) extracts of leaves showed the highest antioxidant activity against DPPH radical. High antioxidant activity of these extracts is due to the presence of flavonoids and phenolic acids [Table 1].
Only EtOAc extract of leaves showed cytotoxicity at high concentrations (500 and 1000 μg/ml) [Table 2]. Extracts with a high content of phenolic acids, methanolic extract of seeds and aqueous extracts of seeds and roots, exhibited neuroprotective effect on hydrogen peroxide induced apoptosis in PC12 cells. DCM and EtOAc extracts showed no neuroprotective activity [Table 3].
DCM and EtOAc extracts of leaves and EtOAc extract of roots showed antiacetylcholinesterase activity by TLC. Three active compounds were detected: one in DCM extract of leaves (Fr=0.61), two in EtOAc extract of leaves and roots (Fr=0.30 and 0.59) [Figure 1].
| Conclusion|| |
Extracts of Galeopsis ladanum L. showed antioxidant activity against DPPH, neuroprotective effect in PC12 cells and antiacetylcholinesterase activity by TLC. These results suggest that Galeopsis ladanum L. may be a potential candidate for the screening of new multipotent compounds for the treatment of Alzheimer′s Disease.
| References|| |
|1.||Rice-Evans C.A. and Packer L. , Flavonoids in Health and Disease . ( Marcel Dekker Inc , New York , 2003 ) |
|2.||Guan S. , Bao Y.M. , Jiang B. and An L.J. Protective effect of protocatechuic acid from Alpinia oxyphylla on hydrogen peroxide-induced oxidative PC12 cell death . Eur J Pharmacol . 538 : 73 - 79 ( 2006 ). |
|3.||Kanga X. , Chena J. , Xub Z. , Lia H. and Wang B. Protective effects of Ginkgo biloba extract on paraquat-induced apoptosis of PC-12 cells . Toxicol in Vitro . 21 : 1003 - 1009 ( 2007 ). |
|4.||Li Y. , Shi W. , Li Y. , Zhoub Y. , Hua X. , Song C. , Ma H. , Wang C. and Li Y. Neuroprotective effects of chlorogenic acid against apoptosis of PC-12 cells induced by methylmercury . Environ Toxicol Pharm . 26 : 13 - 21 ( 2008 ). |
|5.||Brόhlmann C. , Martson A. , Hostettmann K. , Carrupt P.A. and Testa B. Screening of Non-Alkloidal Natural compounds as Acetylcholinesterase Inhibitors . Chem Biodivers . 1 : 819 - 829 ( 2004 ). |
|6.||Houghton P.J. , Ren Y. and Howes M. Acetylcholinesterase inhibitors from plants and fungi . J Nat Prod Rep . 23 : 181 - 199 ( 2006 ). |
|7.||Urbain A. , Marston A. , Sintra Grilo L. , Bravo J. , Purev O. , Purevsuren B. , Batsuren D. , Reist M. , Carrupt P.A. and Hostettmann K. Xanthones from Gentianella amarella ssp. acuta with Acetylcholinesterase and Monoamine Oxidase Inhibitory Activities . J Nat Prod . 71 : 895 - 897 ( 2008 ). |
|8.||Tyunnikova N.V. , Budantsev A.L. and Shavarda A.L. [ The principle biological active substances in the genus Galeopsis L. species ]. Rastitel'Nye Resursy . 40 : 154 - 171 ( 2004 ). |
|9.||Tomαs-Barberαn F.A. , Gil M.I. , Ferreres F. and Tomαs-Lorente F. Correlations between flavonoid composition and infrageneric taxonomy of some european Galeopsis species . Phytochemistry . 30 :. 3311 - 3314 ( 1991 ). |
|10.||Aparicio R. , Oρate J.M. , Arizcun A. , Αlvarez T. , Alba A. , Cuende J.I. and Mirσ M. [ Epidemic rhabdomyolysis due to the eating of quail. A clinical, epidemiological and experimental study ]. Med Clin . 112 :. 143 - 146 ( 1999 ). |
|11.||Jung M. and Park M. Acetylcholinesterase Inhibition by Flavonoids from Agrimonia pilosa . Molecules . 12 : 2130 - 2139 ( 2007 ). |
|12.||Loizzo M.R. , Tundis R. , Menichini F. , Bonesi M. , Statti G.A. , Deguin B. , Tillequin F. , Menichini F. and Houghton P. Acetyl-cholinesterase Inhibition by Extracts and Isolated Flavones from Linaria reflecxa Desf. ( Scrophulariaceae ) . J Nat Prod Com . 2 : 759 - 763 ( 2007 ). |
|13.||Oinonen P. , Jokela J.K. , Hatakka A.I. and Vuorela P.M. Linarin, a selective acetylcholinesterase inhibitor from Mentha arvensis . Fitoterapia . 77 : 429 - 434( 2006 ). |
|14.||Wagner H. and Bladt S. Plant Drug Analysis: A Thin Layer Chromatography Atlas . ( Springer-Verlag , Berlin Heidelberg , 1996 ). |
|15.||Price M.L. and Butler . Rapid visual estimation and spectrophotometric determination of tannin content of Sorghum grain . J Agr Food Chem . 25 : 1268 - 1273 ( 1977 ). |
|16.||Blois M. Antioxidant determination by the use of a stable free radical . Nature . 181 : 1199 - 1200 ( 1958 ). |
|17.||Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays . J Immunol Methods . 65 : 55 - 63 ( 1983 ). |
|18.||Naval M.V. , Gσmez-Serranillos M.P. , Carretero M.E. and Villar A.M. Neuroprotective effect of a ginseng ( Panax ginseng ) root extract on astrocytes primary culture . J Ethnopharmacol . 112 : 262 - 270 ( 2007 ). |
|19.||Ellman G.L. , Courtney K.D. , Andres V. and Feather-Stone R.M. A new and rapid colorimetric determination of acetylcholinesterase activity . Biochem Pharmacol . 7 : 88 - 95 ( 1961 ). |
|20.||Rhee I.K. , Van Rijn R.M. and Verpoorte R. Qualitative determination of false-positive effects in the acetylcholinesterase assay using thin layer chromatography . Phytochem Analysis . 14 : 127 - 131 ( 2003 ). |
[Table 1], [Table 2], [Table 3]
|This article has been cited by|
||Neuroprotective potential of phytochemicals
| ||Kumar, G.P. and Khanum, F. |
| ||Pharmacognosy Reviews. 2012; 6(12): 81-90 |
||Acetylcholine and memory-enhancing activity of Ficus racemosa bark
| ||Ahmed, F. and Narendra Sharath Chandra, J.N. and Manjunath, S. |
| ||Pharmacognosy Research. 2011; 3(4): 246-249 |
||Negative evidence for stachydrine or galeopsis ladanum L. seeds as the causal agents of coturnism after quail meat ingestion
| ||Uriarte-Pueyo, I., Goicoechea, M., Gil, A.G., De Cerain, A.L., De Munain, A.L., Calvo, M.I. |
| ||Journal of Agricultural and Food Chemistry. 2009; 57(22): 11055-11059 |