Chem. Res. Toxicol. 2006, 19, 739-744
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Articles Photochemistry and Photocytotoxicity of Alkaloids from Goldenseal (Hydrastis canadensis L.). 2. Palmatine, Hydrastine, Canadine, and Hydrastinine J. J. Inbaraj, B. M. Kukielczak, P. Bilski, Y.-Y. He, R. H. Sik, and C. F. Chignell* Laboratory of Pharmacology and Chemistry, National Institute of EnVironmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 ReceiVed December 22, 2005
Goldenseal is an herb that is widely used in dietary supplements, eye washes, and skin lotions. The presence of Goldenseal root powder in dietary supplements and the topical application of Goldenseal preparations raise the possibility that an adverse phototoxic reaction may result from an interaction between its constituent alkaloids and light in exposed tissues. We have previously shown that berberine, the major alkaloid in Goldenseal powder, in combination with UVA causes DNA damage and cell death in HaCaT keratinocytes [(2001) Chem. Res. Toxicol. 14, 1529]. We have studied the photochemical and photobiological properties of four minor alkaloids found in Goldenseal, namely, hydrastine, palmatine, canadine, and hydrastinine. UVA radiation of palmatine in aqueous solutions generated no 1O2, but in CH2Cl2, copious amounts of 1O2 were detected (Φ ) 0.2). Palmatine also photogenerated oxygen-centered radicals, •OH and O2•- in aerated aqueous buffer and acetonitrile, respectively, as detected by the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). In nitrogen-sparged acetonitrile containing DMPO, we observed the neutral palmatine radical formed by one-electron reduction. UVA irradiation (4 J/cm2) of HaCaT keratinocytes in the presence of palmatine (50 µM) resulted in a 50% decrease in cell viability but no DNA damage as measured by the comet assay. UVA irradiation of hydrastine, hydrastinine, or canadine (50 µM) did not cause DNA damage or cell death in keratinocytes. Although palmatine is photoactive, it is present in such small amounts in Goldenseal root powder that the phototoxicity of the herb is most likely due to berberine, the major constituent alkaloid. Introduction Preparations derived from the dried root or rhizome of Goldenseal (Hydrastis canadensis L.) can be found in dietary supplements, eardrops, feminine-cleansing products, cold/flu remedies, allergy preparations, laxatives, and digestive aids (1). Goldenseal has also been used to treat wounds and ulcers, as well as skin and eye ailments (2). When applied to the skin, Goldenseal is thought to possess slight antiseptic, astringent, and hemostatic qualities (2). The topical application of Goldenseal raises the possibility that an adverse phototoxic reaction may result from an interaction between the constituent alkaloids and light. Furthermore, photosensitivity has been recently reported in a patient taking a dietary supplement containing Goldenseal (3). We have previously shown that berberine, the major alkaloid in Goldenseal root powder, in combination with UVA1 (320-400 nm wavelengths), causes DNA damage and cell death in HaCaT keratinocytes (4). In addition to berberine, other alkaloids are also present in Goldenseal, including hydrastine, palmatine, and lesser amounts of canadine and hydrastinine (2, 5-7) (Figure 1 and Table 1). We have therefore 1 Abbreviations: PAL•, neutral palmatine radical; DMEM, Dulbecco’s modified Eagle medium; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; EPR, electron paramagnetic resonance; hfs, hyperfine splitting; MTS, 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt; PBS, phosphate-buffered saline; PBS/glucose, PBS containing 10 mM glucose; UVA, 320-400 nm wavelengths.
10.1021/tx050356u
studied the photochemical and photobiological properties of these minor alkaloid constituents of Goldenseal to determine whether they pose a possible phototoxic threat.
Materials and Methods Berberine, palmatine, hydrastine, hydrastinine, and quinine sulfate were obtained from Sigma Chemical Co. (St. Louis, MO). Canadine (tetrahydroberberine) was supplied by Midwest Research Institute (Kansas City, MO). 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) (Aldrich Chemical Co., Milwaukee, WI) was vacuum distilled and stored at -70 °C until use. Acetonitrile (HPLC grade) was purchased from J. T. Baker (Phillipsburg, NJ). All other chemicals were reagent grade or better. Mitotracker Red dye was obtained from Molecular Probes (Eugene, OR). Absorption and Luminescence Spectra. The absorption spectra of the alkaloids (12.5 µM) dissolved in absolute ethanol were recorded in a 10 mm path length quartz cuvette using an HP diode array 8451 spectrophotometer (Hewlett-Packard Co., Palo Alto, CA). Fluorescence spectra were recorded on an SLM SPC 823SMC 220 spectrofluorimeter (SLM Instruments, Urbana, IL). Fluorescence quantum yields were determined using quinine sulfate as a standard and 300 nm excitation wavelength. The uncertainty in the yields was (5%. Singlet oxygen phosphorescence was detected as described previously (8, 9). Perinaphthenone (Aldrich Chemical Co.) was used as a standard for the determinations of the singlet oxygen quantum yields. The excitation wavelengths were 305 nm for hydrastine and canadine and 366 nm for palmatine and hydrastinine. The integrated areas of the 1O2 phosphorescence
This article not subject to U.S. Copyright. Published 2006 by the American Chemical Society Published on Web 05/12/2006
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Inbaraj et al.
Figure 1. Structures of Goldenseal alkaloids. Table 1. Alkaloid Composition of Goldenseal Root Powder
alkaloid
% by weighta
% by weightb
berberine hydrastine palmatine canadine hydrastinine
2.52 1.38 0.22 0.04 not determined
1.9 1.3 0.1 0.06 trace
a
absorption phototoxic integral potential (% by c 280-500 nm weightb × integral (× 10-6) 280-500 nmc × 10-6) 1.93 0.27 2.30 0.12 2.84
3.66 0.35 0.23 0.0072 not calculated
From ref 5. b From ref 6. c Calculated from Figure 2.
spectra for each compound were corrected for the number of photons absorbed over the appropriate wavelengths and for the quenching of 1O2 by these alkaloids. The differences in absorption of each compound were normalized by correcting the absorption spectrum using the Beer-Lambert law and the transmission profile of the source and the filter used for the irradiation. Time-resolved detection of 1O2 phosphorescence was performed as described elsewhere (8). The uncertainty in the yields was (10%. Electron Paramagnetic Resonance (EPR) Spectra. EPR spectra were recorded using a Varian E-109 Century line spectrometer (Varian Associates, Palo Alto, CA) operating at 9.5 GHz with 100 kHz modulation. Samples were placed in a quartz flat cell and irradiated directly inside the microwave cavity of the spectrometer using a 1 kW Xe arc lamp. Radiation from the lamp was passed through a filter (window glass) to remove wavelengths below 300 nm. Hyperfine coupling constants were obtained by accumulating, simulating, and optimizing spectra on an IBM PC computer using software described elsewhere (10). The correlation coefficients for the optimization were 0.98 or better. The uncertainty in the coupling constant measurements was (0.2 G. Cell Viability. HaCaT keratinocytes, a transformed epidermal human cell line (11), were grown at 37 °C in Dulbecco’s modified Eagle medium (DMEM) medium containing 10% fetal calf serum in an atmosphere of 95% air/5% CO2. For the viability studies, the cells were grown in 96 well dishes (Costar, Corning International, Corning, NY). The medium was removed and replaced by sterile phosphate-buffered saline (PBS)/10 mM glucose containing the alkaloids and incubated for 1 h at 37 °C in the dark in a 95% air/ 5% CO2 atmosphere before exposure to UVA radiation from four
fluorescent PUVA lamps (Houvalite F20T12BL-HO, National Biological Corp., Twinsburg, OH). Fluence was measured using a Goldilux UV meter equipped with a UVA probe (Oriel Instruments, Stratford, CT). After exposure, the PBS containing 10 mM glucose (PBS/glucose) solution was removed and replaced with DMEM medium containing 2% fetal calf serum and the cells were kept in the incubator for 24 h. The medium was removed, the cells were washed (2×) with PBS, and the cell viability was measured after the addition of PBS/glucose with the 3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (CellTiter 96 Aqueous Proliferation Assay; Promega Corp., Madison, WI) monitored at 492 nm using the Spectrafluor Plus (Tecan US, Research Triangle Park, NC) plate reader. DNA Damage Assay. DNA damage was assessed using the alkaline single gel (“comet”) assay (12). Briefly, HaCaT keratinocytes grown to ∼85% confluency were trypsinized, and then, ∼50000 cells were suspended in 100 µL of 1% (w/v) low melting point agarose in PBS, pH 7.4, at 37 °C and immediately pipetted onto a frosted microscope slide that had been precoated with 1% (w/v) normal melting point agarose. Slides were allowed to cool at 4 °C for ∼10 min before a second layer of 100 µL of low melting point agarose was pipetted on top of the previous layer. Again, slides were allowed to cool for ∼10 min at 4 °C. The slides were then immersed in a 25 µM solution of alkaloid dissolved in PBS/ glucose and exposed to 1.7 J/cm2 UVA (vide supra). After exposure, the slides were washed once in cold PBS and immediately put into lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, and 1% (v/v) Triton-X 100, NaOH to pH 10.0) at 4 °C overnight. Following lysing, slides were neutralized for 5 min in 0.4 M TrisHCl, pH 7.5. Slides were then placed in a horizontal electrophoresis tank containing 0.3 M NaOH and 1 mM Na2EDTA, pH 13, to unwind for 20 min before electrophoresis at 25 V constant for 40 min. The slides were washed three times for 5 min each with 0.4 M Tris-HCl, pH 7.5, and then placed in cold EtOH for a minimum of 30 min. Slides were dried and stained with 20 µg/mL of ethidium bromide (Sigma Chemical Co.), and 50 comets/slide were scored using Komet 5.0 (Kinetic Imaging Ltd., Liverpool, United Kingdom). Confocal Microscopy. Keratinocytes were seeded into 35 mm dishes containing a glass coverslip-covered 15 mm cutout (MatTek, Ashland, MA) for live cell microscopy measurement. The next day, cells were exposed to palmatine or hydrastinine (20 µM) in PBS for 1 h at 37 °C and then examined by confocal fluorescence microscopy (Zeiss model 510 confocal laser scanning microscope) using 370 nm excitation and a 400 nm band-pass filter. Cells were colabeled with MitoTracker Red (100 nM, Molecular Probes) to determine whether the alkaloids were located in mitochondria. Hydrastine and canadine were not examined by confocal microscopy because the former has a low fluorescence quantum yield, while the fluorescence of the latter occurs at wavelengths (Table 2) where cellular autofluorescence makes detection difficult.
Results Absorption and Fluorescence Spectroscopy. The absorption spectra of the alkaloids are shown in Figure 2. Because sunlight contains wavelengths as short as 290 nm (Figure 2), all of the alkaloids could potentially absorb solar radiation and cause photodamage when present on or in the skin. However, canadine has weak absorption above 300 nm and is found in such small amounts (Table 1) that it is unlikely to act as a cellular photosensitizer. Although hydrastine also has minimal UVA absorption, its concentration in Goldenseal preparations approaches that of berberine (Table 1) and so it has the potential to cause phototoxicity. Hydrastinine has strong UVA absorption but is present in only trace amounts in Goldenseal. The fluorescence quantum yields and emission maxima of the alkaloids in different solvents are given in Table 2, along with those of berberine for comparison. All are either nonfluo-
Alkaloids from Goldenseal
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Table 2. Fluorescence Quantum Yields (Φflu) and Emission Maxima of Goldenseal Alkaloidsa solvent dichloromethane acetonitrile dioxane a
berberine NDc 0.021 (559 nm) 0.071 (524 nm)b
hydrastine
palmatine
canadine
hydrastinine
0.06 (530 nm) 0.0084 (534 nm) ND
0.37 (541 nm) 0.009 (581 nm) 0.24 (519 nm)
ND 0.173 (337 nm) ND
0.06 (468 nm) 0.274 (474 nm) 0.05 (475 nm)
Activation wavelength, 300 nm. b From ref 4. c ND, not determined.
Figure 2. Absorption spectra of the alkaloids dissolved in absolute ethanol. Also shown for comparison is the solar spectral irradiance.
rescent or weakly fluorescent in water (data not shown). Much stronger fluorescence is observed in organic solvents with palmatine and hydrastinine exhibiting high quantum yields in dioxane and acetonitrile, respectively (Table 2). Previously, we were able to use confocal fluorescence microscopy to show that in HaCaT keratinocytes berberine was located mainly in mitochondria with no accumulation in the nucleus (4). However, of the four minor alkaloids, only palmatine and hydrastinine have fluorescence properties amenable to confocal microscopy (hydrastine is nonfluorescent, and canadine fluorescence is indistinguishable from cellular autofluorescence). The subcellular distribution of palmatine fluorescence coincides with that of Mitotracker, a dye that preferentially stains mitochondria (Figure 3A). This finding is in agreement with the report by Mikes and Dadak (13) that berberine and its derivatives locate in the inner mitochondrial membrane where they are responsive to the energized state of the mitochondrion. Although hydrastinine was also present in mitochondria, it was more uniformly distributed than palmatine (Figure 3B). Photogeneration of Singlet Oxygen. The quantum yields of photosensitized 1O2 generation by the alkaloids and berberine in different solvents are given in Table 3. None of the alkaloids generated 1O2 in water or D2O. Palmatine and hydrastinine, like berberine, have high quantum yields of 1O2 photoproduction in dichloromethane, with lower yields in acetonitrile and dioxane. This suggests that palmatine and hydrastinine may generate 1O2 when present in hydrophobic regions of the cell such as membranes. Recently, Hirakawa and co-workers have detected 1O generated by irradiation of berberine and palmatine bound 2 to calf thymus DNA (14). The 1O2 quantum yield of hydrastine was low in dichloromethane while canadine did not generate 1O in any of the solvents examined. 2 EPR Studies. No EPR spectrum was observed when palmatine was irradiated (λ > 300 nm) in nitrogen-saturated aqueous buffer containing DMPO. Irradiation (λ > 300 nm) of palmatine in aerated aqueous buffer containing DMPO generated the EPR spectrum shown in Figure 4A, which was attributed to the DMPO/•OH adduct (aN ) 15.2 G and aH ) 14.9 G). When we repeated the experiment in the presence of ethanol (0.65 M), which reacts rapidly (15; k ) 2 × 109 M-1 s-1) with the
Figure 3. Confocal laser fluorescence microscopy (370 nm excitation and 400 nm band-pass filter) of palmatine (A) and hydrastinine (B) (green) in HaCaT keratinocytes. MitoTracker Red (red) was used to label mitochondria. Yellow shows colocalization of palmatine or hydrastinine with MitoTracker Red. Scale marker ) 20 µm. Table 3. Quantum Yield of 1O2 Photosensitization (Φso) by Goldenseal Alkaloids solvent dichloromethane acetonitrile dioxane a
berberinea hydrastineb palmatinea canadineb hydrastininea 0.34 0.04 0.04
0.03
