MATERIALS AND METHODS

M. I. GEORGIEV ET AL.

plant origin, was used as positive control. Methanol was used as control to ensure that there was no inhibition of

Reagents and chemicals. Electric eel AChE (Type‐VI‐S, EC  3.1.1.7),  horse  serum  BChE  (EC  3.1.1.8),

galanthamine, acetylthiocholine iodide, butyrylthiocholine chloride, and 5,5´‐dithio‐bis(2‐nitrobenzoic)acid (DNTB), butylated hydroxyanisole and chlorogenic acid were purchased from Sigma (St Louis, MO, USA). Ferrozine was purchased from Sigma‐Aldrich Chemie GmbH (Steinheim, Germany). All chemicals used apart from those mentioned above were of analytical grade.

Plant in vitro material. Devil’s claw cell suspension and transformed root cultures were grown in shake‐flasks as described elsewhere (Homova et al., 2010; Stancheva et al., 2011). To evaluate the anti‐cholinesterase and Fe‐chelating activities we applied a scheme for isolating the bioactive metabolites using low pressure chromato- graphic separation, as described by Georgiev et al. (2010) and Gyurkovska et al. (2011). Crude methanolic extracts from cell biomass and hairy roots were designated as CME‐CS (yield: 46.8%) and CME‐HR (yield: 40.3%), respectively. The phenylethanoid‐containing fractions of cell suspension (PF‐CS) and hairy root cultures (PF‐HR) were obtained after loading the crude extracts to a polyamide 6 column and fractions were eluted with portions of distilled H2O and MeOH‐H2O mixtures (25, 50, 75 and 100% MeOH). Verbascoside (the major phenylethanoid in Devil’s claw cells) was further isolated by applying the dried fractions to Merck Lobar columns (RP‐8 and RP‐18), and eluted with a water/methanol gradient as described by Georgiev et al. (2010) and Gyurkovska et al. (2011).

Determination of AChE and BChE inhibitory activities.

AChE and BChE. Dimethyl sulphoxide (DMSO) was not used to dissolve the samples because of its ability to inhibit cholinesterase enzymes.

The ferric‐reducing antioxidant power test. The ferric‐ reducing antioxidant power of the samples was tested using the assay of Oyaizu (1986). One millilitre of the samples, or chlorogenic acid (at 500 mg/mL) as reference for comparative purposes, was added to 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of potassium ferricyanide (1%). The mixture was then incubated at 50 °C for 20 min, and trichloroacetic acid (10%) was then added. After the mixture was shaken vigorously,  this solution was mixed with 2.5 mL of distilled water and

0.5 mL of FeCl3 (0.1%, w/v). After 30‐min incubation, absorbance was read at 700 nm using a Unico 4802 UV‐ visible double beam spectrophotometer (Dayton, NJ, USA). Increase in absorbance of the reaction indicated higher reducing power of the samples.

Fe+2‐ferrozine test system for iron  chelating.  The ferrous ion‐chelating effect of the samples was esti- mated by Fe+2‐ferrozine test system following the method of Chua et al. (2008). Accordingly, 740 μL of

ethanol and the samples were incubated with 0.05 mL of 2 mM FeCl2 solution. The reaction was initiated by the addition of 40 μL of 5 mM ferrozine solution into the mixture and left standing at ambient temperature for 10 min. The absorbance of the reaction mixture was measured at 562 nm. The ratio of inhibition of ferrozine‐Fe2+ complex formation (I) was calculated as

follows:

(Acontrol −Asample)

The AChE and BChE inhibitory activities of the extracts,

phenylethanoid fractions and pure verbascoside were determined by the modified spectrophotometric method

I

Acontrol

X 100; %

of Ellman et al. (1961), using acetylthiocholine and butyrylcholine chloride as substrates. Acetylthiocholine iodide and butyrylthiocholine chloride were employed as substrates for the reaction. 5,5´‐Dithio‐bis(2‐nitrobenzoic) acid was used for the measurement of the cholinesterase activity. All the other reagents  and  conditions  were the same as described in Senol et al. (2010). In brief,

140 μL of 0.1 mM sodium  phosphate  buffer  (pH 8.0), 20 μL of DTNB, 20 μL of sample solutions and 20 μL of AChE/BChE solution were added using multichannel automatic pipette (Pipetman, Gilson, France) in a 96‐well microplate and incubated for 15 min at 25°C. The reac- tion was then initiated with the addition of 10 μL of acetylthiocholine iodide/butyrylthiocholine chloride. The enzymatic hydrolysis of acetylthiocholine iodide (0.4 mM)/ butyrylthiocholine chloride (0.2 mM) formed thiocholine, which was cleaved using 0.25 mM DNTB into the yellow 5‐thio‐2‐nitrobenzoate, and further monitored at a wavelength of 412 nm utilizing a 96‐well microplate reader (VersaMax, Molecular Devices, ML,  USA). The  mea- surements and calculations were evaluated by using Softmax PRO 4.3.2.LS software. Percentage of inhibition of AChE/BChE was determined by comparison of the rates of reaction of samples and blank samples (ethanol in phosphate buffer pH 8) using the formula (E − S)/E × 100, where E is the activity of enzyme without test sample and S is the activity of enzyme with test sample. Galanthamine (at 100 mg/mL), the anticholinesterase alkaloid drug of

The controls contained only FeCl2 and ferrozine, while butylated hydroxyanisole (BHA; at 100 mg/mL) was used as the reference in this assay.

Statistics. All analyses were run at least in triplicate and expressed as average values ± SEM (standard error mean).

RESULTS AND DISCUSSION

We examined the ability of H. procumbens extracts, fractions and pure verbascoside to inhibit the enzymes AChE and BChE (Table 1). The highest AChE inhibitory activity was displayed by the phenylethanoid‐containing fraction, obtained from transformed root culture (PF‐HR), followed by pure verbascoside and phenylethanoids fraction of cell biomass (PF‐CS). Crude methanolic extracts of both cell suspension (CME‐CS) and trans- formed root (CME‐HR) cultures displayed no AChE inhibitory activity. All positive samples showed weaker AChE inhibition activity than pure galanthamine, used as a reference (Table 1). However, the activity of the phenylethanoid‐containing fraction from H. procumbens hairy roots was several times higher than these reported before for 55 Salvia taxa (Senol et al., 2010). In BChE inhibition assay the activity was PF‐CS > PF‐HR > CME‐ CS > CME‐HR > verbascoside  (Table  1).  It  is  worth

Copyright © 2011 John Wiley & Sons, Ltd.                                                                                                                                                    Phytother. Res. (2011)

mentioning that the  phenylethanoid‐containing  fraction of Devil’s claw cell biomass exhibited ~9% higher BChE inhibitory activity than pure galanthamine. As at a con- centration of < 250 µg/mL the PF‐CS displayed no toxicity (in 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay; Gyurkovska et al., 2011) it could be suggested that the fraction might be a promising BChE inhibitor. Currently, there is no information available for AChE‐ and BChE‐inhibitory activities, neither for Devil’s claw plants nor for their active constituents (at least in the SCOPUS database: accessed 6 December 2010).

The crude methanolic extracts  from Devil’s claw cell and hairy roots biomass did not show cholines- terase inhibitory activity, although they contained phenylethanoids in small concentrations. Therefore, enrichment of the bioactive substances in the extracts is essential if they are to be used as cholinesterase inhibitors. Furthermore, there are some reports de- scribing the neuroprotective properties of verbascoside (Wang et al., 2009).

The FRAP assay uses antioxidants as reductants in a redox‐linked colorimetric method (Benzie and Strain, 1999). In the FRAP assay the highest activity was displayed by verbascoside, followed by phenylethanoid‐ containing fractions and crude methanolic extracts of cell biomass and transformed roots (Table 2). The FRAP values of the most potent samples (e.g. verbascoside, PF‐CS and PF‐HR) were ~36–40% lower than those of reference chlorogenic acid.

In contrast to the FRAP assay, Fe‐chelating activity was exhibited only for crude methanolic extracts (Table 2). The Fe‐chelating activity of crude methanolic

extract from Devil’s claw cell biomass and transformed root was significantly higher compared with pure butylated hydroxyanisole, used as positive standard (~1.5 times  and ~2 times higher, respectively). How- ever, phenylethanoid fractions and pure verbascoside isolated from them displayed no activity (Table 2). Therefore it could be suggested that some compounds, albeit present in minor amounts in crude methanolic extracts, exhibited superior Fe‐chelating capacity.

In conclusion, phenylethanoid fractions, obtained by simply applying one‐step  chromatographic  separation, of Devil’s claw cell biomass and hairy roots mass show potential as anti‐AChE and anti‐BChE agents due  to their strong inhibitory activity. Therefore, it can be suggested that in addition to providing a potent source of antiinflammatory compounds (Gyurkovska et al., 2011) Devil’s claw in vitro systems are attractive sources of powerful cholinesterase inhibitors for the pharmaceutical  industry.

Acknowledgements

The authors express their thanks to Dr G. Kerns (SIAB, Leipzig, Germany) for kindly supplying the Harpagophytum procumbens hairy root and callus culture clones. This research has been supported by a grant (contract number DO‐02‐261/2008) from the National Science Fund of Bulgaria.

Conflict of Interest

The authors have declared that there is no conflict of interest.

Table 2. Ferric‐reducing antioxidant power (FRAP) and Ferric ion‐chelating capacity of extracts, fractions and pure compound of

Harpagophytum procumbens in vitro systems

FRAP (absorbance at 700 nm)a                                                                               Ferric ion‐chelating capacity (% of inhibition)a

aMeans ± SE (n = 3).

NA, no activity; ND, not determined.

Copyright © 2011 John Wiley & Sons, Ltd.                                                                                                                                                   Phytother. Res. (2011)

M. I. GEORGIEV ET AL.

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