versão impressa ISSN 0102-695X
Rev. bras. farmacogn. vol.21 no.4 Curitiba jul./ago. 2011 Epub 17-Jun-2011
Clara GrossoI; Juliana VinholesI; Luís R. SilvaI, Paula Guedes de PinhoII, Rui F. GonçalvesI; Patrícia ValentãoI, Anna K. JägerIII, Paula B. AndradeI
IREQUIMTE/Laboratório de Farmacognosia, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, Portugal
IIREQUIMTE/Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Portugal
IIIDepartment of Medicinal Chemistry, Faculty of Pharmaceutical Sciences University of Copenhagen, Denmark
Capsella bursa-pastoris (L.) Medik. (Brassicaceae) is a wild herb with high nutritional value that can be eaten raw or cooked. A metabolomic study was performed with different extracts of its aerial parts that were tested concerning their antiradical, acetylcholinesterase inhibitory and antibacterial activities. Phenolic compounds were identified and quantified by HPLC-DAD, organic acids and amino acids were determined by HPLC-UV, while free fatty acids and sterols were analysed by GC-ITMS. The vegetal material was rich in kaempferol-3-O-rutinoside (mean value 2247.09 mg/kg of dry plant), quinic acid (95628.00 mg/kg of dry plant), arginine (mean value of 1.18 mg/kg of dry plant), palmitic acid (284.48 mg/kg) and β-sitosterol (28%). The extracts presented a concentration-dependent antiradical activity (against DPPH, O2- and LOO), being most effective against NO (EC25 0.20 µg/mL). In addition, the extracts were also acetylcholinesterase inhibitors and antibacterial active, revealing that, besides the plant's good nutritional value, it presents important biological properties as well.
Keywords: acetylcholinesterase inhibition antibacterial activity, antioxidant activity, Capsella bursa-pastoris, metabolic profile.
Capsella bursa-pastoris (L.) Medik., Brassicaceae, commonly known as shepherd's purse, is a wild plant, whose young leaves and roots have been used as an edible vegetable, eaten raw or cooked in some countries (Zennie & Ogzewalla, 1977; Kweon et al., 1996). The nutritional composition of this species, including minerals, vitamin A, ascorbic acid, proteins, linoleic acid and Ω3 polyunsaturated fatty acids, is considered to be beneficial to human health (Zennie & Ogzewalla, 1977; Guil-Guerrero et al., 1999). Besides this, C. bursa-pastoris has some medicinal properties, being indicated as anti-bleeding, anticancer, antithrombin (Bekker et al., 2002; Goun et al., 2002), wound-healing (Park et al., 2000) and antioxidant agent (Ivanova et al., 2005), as well as for diabetes and fever treatment (Kweon et al., 1996). However, an approach to the chemical composition was not provided by the authors. On the other hand, Park et al. (2000) isolated peptides from the roots that showed activity against several strains of bacteria and fungi, while an isolated flavonoid-O-glucoside revealed to be a superoxide anion radical scavenger (Kweon et al., 1996).
From the chemical point of view, several classes of secondary metabolites have already been found in this species, such as phenolic compounds, mainly flavonoids (Song et al., 2007), alkaloids, namely calystegines (Brock et al., 2006), glucosinolates (Cole, 1976) and saponins (Marquina et al., 1955).
Considering the scarce literature on the metabolic composition and the broad spectrum of activities observed for this species, the aim of this study was to characterize the aerial parts of C. bursa-pastoris, focusing on flavonoids, organic acids, fatty acids, sterols, and amino acids. Subsequently, several in vitro (antioxidant, anti-cholinesterase and antibacterial) assays were carried out.
Materials and Methods
The dried aerial parts of Capsella bursa-pastoris (L.) Medik. (Brassicaceae) purchased in the local market were from a medicinal plants distributor (Morais e Costa & C.ª Lda, Portugal). The identity was confirmed by the authors following the characteristics described in Floras (Coutinho, 1939; Flora Ibérica, 2003). The acquired sample was also compared with an individual occurring in nature. The plant material was powdered (mean particle size lower than 910 µm). Voucher specimen was deposited at Laboratório de Farmacognosia, Faculdade de Farmácia, Universidade do Porto (CBP-AP-032010).
Standards and reagents
Reference compounds were purchased from various suppliers: flavonoid standards were from Extrasynthèse (Genay, France). 1,1-Diphenyl-2-picrylhydrazyl (DPPH), β-nicotinamide adenine dinucleotide (NADH), phenazine methosulphate (PMS), nitroblue tetrazolium chloride (NBT), 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), sulphanilamide, acetylcholinesterase (AChE) (CAS Registry No. 9000-81-1; EC 232-559-3, type VI-s) from electric eel (Electrophorus electricus), acetylthiocholine iodide (ATCI), Tris-HCl, hexane, 2-propanol, L-amino acid kit, organic acids and sterols standards were purchased from Sigma (St. Louis, MO, USA). N-(1-Naphthyl) ethylenediamine dihydrochloride, ferrous sulphate heptahydrate, ethanol, methanol, ethyl ether, dichloromethane and chloride, sulphuric and formic acids were obtained from Merck (Darmstadt, Germany). Sodium nitroprussiate dehydrate (SNP) and ascorbic acid were purchased from Sigma-Aldrich (Steinheim, Germany). Linoleic acid was from BDH chemicals, Ltd. (Poole, England). Trichloromethane, sodium sulphate anhydrous and isooctane were purchased from Panreac Química SA (Barcelona, Spain). Potassium hydroxide was from Pronalab (Lisboa, Portugal) and boron trifluoride (BF3) 10% methanolic solution, dabsyl chloride and fatty acid methyl esters (FAME kit were purchased from Supelco (Bellafonte, PA, USA)). Methyl jasmonate (internal standard) was from SAFC (St. Louis, USA). Mueller Hinton broth (MHB) and Mueller Hinton agar (MHA) were purchased from Liofilchem (Roseto degli Abruzzi, Italy).
The extraction of aerial parts of C. bursa-pastoris was performed with methanol (MeOH), and methanol:water (1:1) mixture (MeOH/H2O) (1 g/100 mL), applying the following steps: sonication (1 h), stirring maceration at room temperature (200 rpm, 16 h), plus sonication (1 h), filtration and evaporation under reduced pressure.
For the identification of C-heterosides 2 N HCl was added to methanolic extract (250 µL) and heated at 100 ºC (1 h). The hydrolysed extract was applied in a Chromabond C18 column (500 mg sorbent mass/6 mL reservoir volume), conditioned with methanol (5 mL) and acid water (5 mL, pH 2 with HCl), and eluted with methanol (5 mL). The eluate was dried, redissolved in methanol (250 µL), and analysed (20 µL) by HPLC.
HPLC/DAD analysis and quantification
The extracts were analysed on an analytical HPLC unit (Gilson), using a previously described procedure (Oliveira et al., 2009). Detection was achieved with a Gilson diode array detector, spectral data were collected in 200-400 nm range, and chromatograms were recorded at 350 nm. The data were processed on an Unipoint System software (Gilson Medical Electronics, Villiers le Bel, France). Phenolic compounds quantification was achieved by external standards method. Quercetin-6-C-glucoside (not commercially available) was quantified as quercetin-3-O-glucoside, being all other compounds quantified by their correspondent standard.
Organic acids were extracted from 1 g of raw material with H2SO4 (50 mL, 0.01 N), under stirring (200 rpm, 30 min), followed by filtration and evaporation. The resulting extract was redissolved in H2SO4 (1 mL, 0.01 N) and subjected to analysis (20 µL).
Organic acids were analysed on an analytical HPLC unit (Gilson), using an ion exclusion Nucleogel Ion 300 OA column (300 × 7.7 mm) (Germany), with a column heating device (30 ºC) (Oliveira et al., 2009). Elution (70 min) was carried out isocratically, using H2SO4 0.01 N (0.2 mL/min). Detection was performed with a Gilson UV-Vis detector at 214 nm. The organic acids quantification was achieved relatively to external standards.
Forty milligrams of each extract (MeOH and MeOH/H2O) of C. bursa-pastoris was dissolved in HCl (400 µL, 0.1 M). The derivatization procedure was described previously by Oliveira et al. (2008).
Dabsyl derivatives of free amino acids were analysed on a Gilson HPLC unit, using a C18 Spherisorb ODS2 column (25.0 cm × 0.46 cm; 5 µm particle size) from Waters (Ireland), applying the same conditions of Oliveira et al. (2008).
The extraction and derivatization procedures were performed according to Ribeiro et al. (2009).
FAME quantification was performed by external standards calibration curves obtained by GC-ITMS using the conditions described by Ribeiro et al. (2009). One microliter of the extract was analysed in Full Scan mode. Identification of compounds was achieved by comparison of their mass spectra and retention times with those from pure standards (FAME kit purchased from Supelco (Bellafonte, PA, USA), analysed under the same conditions, and with NIST 05 MS Library Database.
The sterols extraction of aerial parts of C. bursa-pastoris was performed with dichloromethane (DCM), in a ratio of 1 g/100 mL, using the following steps: sonication (1 h), stirring maceration at room temperature (16 h), plus sonication (1 h). After filtration the extracts were evaporated under reduced pressure, redissolved in DCM and analysed by GC-ITMS.
The sterols were estimated by their relative abundance from their total chromatographic area. The injector port was heated to 250 ºC, and injections were performed in split mode (1/40), the oven temperature was set at 40 ºC (for 1 min), then increasing 2 ºC/min to 220 ºC and held for 30 min. All mass spectrometer parameters were kept as mentioned in Ribeiro et al. (2009) with the exception of m/z mass range (50 to 1000). The compounds identification was achieved by comparing their retention indices and mass spectra with those of authentic reference compounds, and with NIST 05 MS Library Database.
The free radical-scavenging activity was determined in a Multiskan Ascent plate reader (Thermo Electron Corporation), by monitoring the decrease of absorbance of DPPH, according to Oliveira et al. (2009).
Superoxide anion (O2-) assay
O2- was generated by the NADH/PMS system, as referred in Oliveira et al. (2009).
Nitric oxide (NO) assay
The antiradical activity was determined in a Multiskan Ascent plate reader (Thermo Electron Corporation), using a previously described procedure (Oliveira et al., 2009).
Lipid peroxidation (LOO) inhibition
Lipid peroxyl radical (LOO) was generated according to the method described by Choi et al. (2002). The detection of conjugated dienes was measured with slight modifications to the method reported by Shimasaki (2000). The absorbance at 233 nm was measured in a spectrophotometer (Helios α, Unicam) at room temperature.
Acetylcholinesterase (AChE) inhibitory activity
The inhibition of AChE activity was determined spectrophotometrically in a Multiskan Ascent plate reader (Thermo Electron Corporation), based on Ellman's method, as previously reported (Oliveira et al., 2009). Physostigmine was used as a positive control (data not shown).
Nine bacteria species were used: Staphylococcus aureus (ATCC 20231), Staphylococcus epidermidis (ATCC 20044), Micrococcus luteus (ATCC 20030), Enterococcus faecalis (ATCC 20477), Bacillus cereus (ATCC 31), Proteus mirabilis (ATCC 4479), Escherichia coli (ATCC 30083), Pseudomonas aeruginosa (ATCC 50071) and Salmonella typhimurium (ATCC 43971). All cultures were obtained from the Department of Microbiology, Faculty of Pharmacy, Porto University, Portugal. The stock culture was maintained on MHA at 4 ºC.
Preparation of inoculums
Bacterial inoculums were prepared by growing cells in MHB for 24 h at 37 ºC. Cell suspensions were diluted with sterile MHB to provide initial cell counts of about 106 CFU/mL.
Minimum inhibitory concentration (MIC)
The minimum inhibitory concentration of C. bursa-pastoris extracts was assessed by the twofold serial dilution method as described by Taveira et al. (2010). The MIC was determined as the lowest concentration of the raw material inhibiting the visible growth of the test culture on the microplate.
Results and Discussion
The MeOH and MeOH/H2O extracts of C. bursa-pastoris analysed by HPLC-DAD allowed the identification of five flavonoids, namely, quercetin-6-C-glucoside, quercetin-3-O-glucoside, kaempferol-3-O-rutinoside, quercetin, and kaempferol (Table 1, Figure 1a). Kaempferol-3-O-rutinoside was the main phenolic in both MeOH and MeOH/H2O extracts, representing 65 and 51 % of total determined compounds, respectively (Table 1). The presence of the C-heteroside described by Song et al. (2007), quercetin-6-C-glucoside, was confirmed in both extracts by HPLC/DAD analysis of the hydrolysed extracts (Figure 1b). The hydrolysis is just able to cleave the O-glycosyl bond, being C-bond resistant. Only one compound remained after samples' treatment and quercetin and kaempferol contents increased. Therefore, it was assumed that the C-heteroside was a flavonol derivative, most probably quercetin-6-C-glucoside reported before.
Six compounds were identified in C. bursa-pastoris (Figure 2) acidic extract, in concentrations ranging from 8.02 to 95628.00 mg/kg (Table 2). Among the identified compounds only oxalic acid was previously described in this species (Guil-Guerrero et al., 1999). Quinic, malic and citric acid were responsible for 97% of the total organic acids content, being the first one the most representative (48%).
A total of eighteen amino acids (AA) were found in MeOH (Figure 3) and MeOH/H2O C. bursa-pastoris extracts, but their AA profile was distinct. Threonine and isoleucine were found only in MeOH, while glutamic acid, asparagine and tryptophan were just identified in MeOH/H2O extract. Arginine and tyrosine were the main amino acids in both extracts, where the first one represents more than 50% and the second 30% of the total amino acid content (Table 3). As far as we know, this is the first report on amino acids in this species.
Thirteen fatty acids (FA) were quantified in C. bursa-pastoris extract. From these, heptadecanoic acid is reported for the first time. As can be seen in Table 4 and Figure 4, the FA compounds found exhibit C12:0 to C20:0 structures, with saturated (8), monounsaturated, (4) and polyunsaturated (1) bonds. Among these, palmitic acid (hexadecanoic acid, C16:0) was the main compound, representing 52% of the FA composition, followed by stearic acid and oleic acid, both representing approximately 10% of the total FA found. These results were in accordance with the study performed on the aerial parts of C. bursa-pastoris (Bekker et al., 2002), where palmitic acid and oleic acid represented 50 and 12%, respectively.
Several sterols were identified in C. bursa-pastoris dichloromethane extract (Figure 5), and their relative contents are shown in Table 5. β-Sitosterol, the only sterol described so far, was the main compound; thus, its abundance was considered as 100% and the other compounds reported against it. The contribution of the other compounds followed the order campesterol (38%) > stigmasta-4-en-3-one (14%) > cholesterol (7%) > stigmasterol (6%) > cholest-5-en-3-one (5%) > ergosta-4,6,8(14),22-tetraen-3-one (3%) > lupeol (2%) > stigmasta-3,5-dien-7-one (2%).
Plant extracts with antiradical activity are of great importance when an uncontrolled production of reactive species occurs in the organism, and the endogenous antioxidants are not capable to overcome their deleterious effect. The search of new bioactive compounds from natural sources has been motivated by the concern about the safety of synthetic antioxidants (Williams et al., 1999). A concentration-dependent pattern was observed in all antioxidant assays (Figure 6a-d, Table 6), which is in accordance with results obtained for other plants belonging to the Brassicaceae family (Orhan et al., 2009). MeOH/H2O extract was more effective against DPPH, O2- and NO, while MeOH was more active for LOO. Extracts that efficiently act against O2- and NO radicals are considered very important, once they can prevent the formation of other deleterious radicals, like peroxynitrite (Pacher et al., 2007).
The main difference observed between the extracts composition is in the content of quercetin and its derivatives, present in higher amounts in the MeOH/H2O extract. The radical scavenging activity of flavonoids is partially related with the catechol group in the B-ring; therefore, quercetin activity is more pronounced than that of kaempferol (Ji & Zhang, 2006). However we should also consider the presence of other bioactive compounds, once MeOH extract was more effective as LOO scavenger. For instance, the presence of compounds, such as i) quinic acid, which is described as antioxidant (Pero et al., 2009), ii) stearic acid, which is involved in the inhibition of lipid peroxidation and in the promotion of antioxidant enzymes activity, like Cu/Zn superoxide dismutase (SOD) and catalase (Wang et al., 2007), and iii) oleic acid, as well as other monounsaturated fatty acids, which are involved in the reduction of the susceptibility of LDL to oxidation (Tsimikas et al., 1999), should be taken into account. The presence of a Ω6 fatty acid (linoleic acid), even in low concentrations, seems to be very important because this compound is involved in diminishing coronary disease risk (WHO, 2003). Ω3 Fatty acids, also biologicaly important, were not detected in C. bursa-pastoris analysed extract. Moreover, the presence of phytosterols can inhibit the dietary and biliary cholesterol uptake (Klingberg et al., 2008).
All over the world, 24 million people suffer from dementia, with Alzheimer's disease (AD) being the most common cause in the elderly (WHO, 2006). It is assumed that the dysfunction of cholinergic neurotransmission in the brain contributes to the relevant cognitive decline in AD. The loss of cholinergic cells is accompanied by the loss of the neurotransmitter acetylcholine, thus, one of the most accepted strategies in AD treatment is the use of cholinesterase inhibitors (de Paula et al., 2009; Vinutha et al., 2007). Under the assay conditions, MeOH and MeOH/H2O extracts exhibited high acetylcholinesterase inhibitory capacity (Table 6, Figure 7). Our results are in agreement with the AChE inhibition observed for other Brassicaceae (Local Food-Nutraceuticals Consortium, 2005; Orhan et al., 2009). These species showed an AChE inhibition of 10-19% at a concentration of 0.1 mg/mL, which correspond to the same range of inhibition observed for the C. bursa-pastoris MeOH extract.
As observed for LOO inhibition, MeOH extract was more active than MeOH/H2O over AChE. As above, we have to consider the overall interaction between the different classes of compounds identified in this species (phenolic compounds, organic acids, amino acids, fatty acids, and sterols).
In a general way, the results obtained in the performed assays revealed that these extracts are efficient free radical scavengers and moderate inhibitors of AChE as well.
The uncontrolled use of antibiotics can cause microorganisms resistance. Therefore, actions must be taken to develop new antimicrobial agents from natural sources (Chandrasekaran et al., 2008). In order to evaluate the antibacterial potential of C. bursa-pastoris, MeOH, MeOH/H2O and dichloromethane extracts were screened for activity against five Gram-positive and four Gram-negative bacteria. The MIC was calculated to evaluate the extracts' effects. As can be observed in Table 7, the MICs obtained for MeOH and MeOH/H2O extracts were lower than those of DCM. In addition, Gram-positive bacteria were more susceptible than Gram-negative ones.
In a general way, the MeOH/H2O extract was more effective, with MICs bellow 32.00 mg/mL for all Gram-positive bacteria, while for methanol this was observed only for S. epidermidis and M. luteus (Table 7). Since immunocompromised patients are more vulnerable to these two microorganisms (Prescott et al., 1996) extracts that are active against them can be of great importance. The MICs observed for MeOH/H2O extract against S. aureus, E. faecalis and B. cereus were lower than those observed for MeOH extracts. The effective action against these pathogenic microorganisms is also important, as they are involved, for instance, in the origin of urinary tract infections and endocarditis (E. faecalis) (Prescott et al., 1996).
Previous studies already indicated that Gram-positive bacteria appear to be more sensitive to the action of many natural extracts (Dorman & Deans, 2000), which is in agreement with our results.
The different degree of activity observed for C. bursa pastoris extracts can be ascribed to the presence of several classes of compounds. In particular, the higher antibacterial effect of MeOH/H2O and MeOH can be related, at least partially, to the presence of phenolic compounds. Thus, the more pronounced antibacterial effect observed for MeOH/H2O extracts can be explained by the higher content of quercetin that is already known by its ability to increase the membrane permeability (Cushnie & Lamb, 2005).
The identification and quantification of primary (organic acids, amino acids and fatty acids) and secondary (phenolic compounds, and sterols derivatives) metabolites in C. bursa-pastoris, as well as the screening of a variety of biological activities, was achieved. Twenty seven compounds are reported for the first time in this species. Besides the good nutritional value already described, this species revealed to be an interesting source of bioactive compounds. We can highlight the extracts' antioxidant and antibacterial activities, suggesting that they may be interesting not only for human health but also as food additive. Moreover the AChE inhibitory activity, an important factor in Alzheimer's disease, may extend the use of C. bursa-pastoris for pharmaceutical applications.
Clara Grosso thanks the Fundação para a Ciência e a Tecnologia for the Post-Doc fellowship (SFRH/BPD/63922/2009)
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Paula B. Andrade
REQUIMTE/Laboratório de Farmacognosia, Departamento de Química, Faculdade de Farmácia, Universidade do Porto
R. Aníbal Cunha, 164, 4050-047 Porto, Portugal
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Received 10 Dec 2010
Accepted 15 Feb 2011