Chem. Res. Toxicol. 2001, 14, 1529-1534
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Photochemistry and Photocytotoxicity of Alkaloids from Goldenseal (Hydrastis canadensis L.) 1. Berberine J. J. Inbaraj, B. M. Kukielczak, P. Bilski, S. L. Sandvik, 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 May 3, 2001
Goldenseal is an herb which is widely used for many medical applications such as in eyewashes and skin lotions and which is currently undergoing testing by the National Toxicology Program. The main alkaloid constituent of Goldenseal is berberine. The topical application of Goldenseal or berberine to the skin or eyes raises the possibility that an adverse phototoxic reaction may result from an interaction between the alkaloid and light. We have therefore studied the photochemistry of berberine in different solvents and its phototoxicty to HaCaT keratinocytes. Irradiation of berberine in aqueous solutions does not generate 1O2, but in CH2Cl2, 1O2 is produced with a quantum yield φ ) 0.34. With the aid of the electron paramagnetic resonance (EPR) spin trapping technique and 5,5-dimethyl-1-pyrroline N-oxide (DMPO), we have detected oxygen-centered radicals photogenerated by berberine in water and acetonitrile. In the latter solvent and in the absence of oxygen, the neutral berberine radical formed by one electron reduction was observed. Methanol radicals were detected by EPR in water/alcohol low-temperature glasses irradiated in the berberine long-wavelength absorption band. In such alcoholic glasses, we have also detected an EPR signal from the berberine triplet at 77 K, in contrast to aqueous glasses where neither triplet nor radicals were detectable. Our data show that, although a weak photosensitizer in water, berberine is able to produce both 1O and radical species in a nonpolar environment. UVA irradiation of HaCaT keratinocytes 2 in the presence of 50 µM berberine resulted in an 80% decrease in cell viability and a 3-fold increase in DNA damage as measured by the Comet assay. These findings suggest that exposure to sunlight or artificial light sources emitting UVA should be avoided when topical preparations derived from Goldenseal or containing berberine are used.
Introduction The dried root or rhizome of Goldenseal (Hydrastis canadensis L.) has been used to treat wounds and ulcers, as well as skin and eye ailments (1). When applied topically, Goldenseal is thought to possess slight antiseptic, astringent, and hemostatic qualities (1). The main alkaloid constituents of Goldenseal are berberine and (-) hydrastine with minor amounts of canadine and palmatine (1) (Figure 1). Berberine itself has been employed to treat skin diseases, including psoriasis (2), and eye infections (3). The use of berberine as a fluorescent stain for cells (4), dermosomes (5), and energized mitochondria (6) indicates that there is a strong interaction with cellular components. Thus, the topical application of Goldenseal or berberine to the skin or eyes raises the possibility that an adverse phototoxic reaction may result from an interaction between the alkaloid and light. We have therefore studied the photochemistry of berberine in different solvents and its phototoxicty to HaCaT keratinocytes.
Materials and Methods Berberine chloride was from Sigma Chemical Co. (St. Louis, MO) and used as received. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO)1 (Aldrich Chemical Co., Milwaukee WI) was vacuum * To whom correspondence should be addressed.
10.1021/tx0155247
Figure 1. Structures of Goldenseal alkaloids. distilled and stored at -70 °C until use. 3,3,5,5-Tetramethyl1-pyrroline N-oxide (TMPO) and phenyl-tert-butyl nitrone were
This article not subject to U.S. Copyright. Published 2001 by the American Chemical Society Published on Web 10/18/2001
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from Aldrich and used as received. Methanol and acetonitrile (HPLC grade) were purchased from J. T. Baker (Phillipsburgh, NJ). All other chemicals were of reagent grade or better. Superoxide dismutase was from Boehringer Mannheim, Germany. Absorption spectra were recorded using an HP diode array 8451 spectrophotometer (Hewlett-Packard Co., Palo Alto, CA). Fluorescence and phosphorescence spectra were recorded on an SLM SPC 823-SMC 220 spectrofluorimeter (SLM Instruments, Urbana, IL). Singlet oxygen phosphorescence was detected as described previously (7, 8). Phenalenone (Aldrich Chemical Co., Milwaukee WI) was used as standard for the determinations of the singlet oxygen quantum yields. The integrated areas of the 1O phosphorescence spectra for each compound were corrected 2 for the number of photons absorbed over the appropriate wavelengths and for the quenching of 1O2 by these antibiotics. 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). EPR spectra were recorded using a Varian E-109 Century line spectrometer 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 couplings were obtained by accumulating, simulating, and optimizing spectra on an IBM PC computer using software described elsewhere (9). For the low-temperature EPR studies berberine was dissolved in 50% spectroscopic-grade methanol (Sigma Chemical Co.). Samples were maintained at 77 K, within the microwave cavity in a liquid nitrogen suprasil dewar (Wilmad Glass, Bueno, NJ), and irradiated directly in the cavity with a 200 W mercury arc lamp. Typical spectrometer parameter settings were power, 10dB; modulation amplitude, 1 G; modulation frequency, 100 Hz; time constant, 0.5 s. Cell Viability. HaCaT keratinocytes, a transformed epidermal human cell line (10), were grown at 37 °C in Epilife medium (Cascade Biologics Inc., Portland, OR) in an atmosphere of 95% O2/5% 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 PBS containing berberine at different concentrations and incubated for 1 h at 37 °C in the dark in a 95% O2/5% CO2 atmosphere before exposure to UVA radiation from 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 irradiation the PBS was removed and replaced with Epilife medium. Cell viability was determined using the formazan assay (CellTiter96 Aqueous Non-Radioactive Cell Proliferation Assay, Promega Corp., Madison, WI). DNA Damage Assay. Keratinocytes grown in plastic dishes to ∼80% confluence were exposed to berberine and UVA as described for the viability assay (vide supra) except that just before UVA exposure the berberine solutions were replaced with PBS buffer. After exposure, the cells were trypsinized, centrifuged, and counted, and then DNA damage was assessed using the alkaline single gel (“comet”) assay (11). After electrophoresis, fluorescent images of the nuclei stained with ethidium bromide were captured with a video camera and digitized using the Matrox Meteor II interface (Matrox Image, Quebec, Canada) to a PC controlled by the Matrox Inspector program. A script written in BASIC was used to measure the comet moment (% of pixel intensity in the tail x distance from head to tail in microns) from at least 50 cells in each group. 1 Abbreviations: BER•, berberine radical; DMPO, 5,5-dimethyl-1pyrroline N-oxide; EPR, electron paramagnetic resonance; hfs, hyperfine splitting; GSH, reduced glutathione; PBN, phenyl-tert-butyl nitrone; PBS, phosphate buffered saline; SOD, superoxide dismutase; TMPO, 3,3,5,5-tetramethyl-1-pyrroline N-oxide.
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Figure 2. Absorption, fluorescence and phosphorescence of berberine. The absorption spectrum of berberine (50 µM) in water (Aw) and ethanol (Ae). The fluorescence emission spectrum of berberine (40 µM) in water (Fw) and ethanol (Fe). The phosphorescence of berberine in frozen (77 K) ethanol (Pe). Inset: lifetime of berberine phosphorescence. Table 1. Selected Spectral, Photochemical, and Photophysical Properties of Berberine solvent
abs maxa (nm)
fl maxb (nm)
ΦFc
Φsod
CH2Cl2 toluene 1,4-dioxane EtOH MeCN propylene carbonate water (D2O)
436 ( 2 426 ( 2 426 ( 2 428 ( 2 428 ( 2 428 ( 2 422 ( 2
ndg nd 524 ( 3 546 ( 3 552 ( 3 556 ( 3 556 ( 3
nd nd 0.071 0.036 0.025 0.007 0.000 47
0.34 0.044e 0.042 nd 0.038 nd 300 nm) in nitrogen saturated aqueous buffer containing DMPO. Irradiation (λ > 300 nm) of berberine in aerated aqueous buffer containing DMPO generated the EPR spectrum shown in Figure 5A. Analysis of the spectrum indicated the presence of two components. The first was attributed to the DMPO/•OH adduct (aN ) 15.2, aH ) 14.8G) while the second (1, aN ) 16.5G aH ) 23.3 G) was an unidentified carbon-centered adduct. The intensity of the DMPO/ • OH adduct was drastically reduced upon the addition of SOD (Figure 5B), suggesting that this radical was partially derived from decomposition of the DMPO/O2•adduct (14) rather than reaction of DMPO with the •OH radical. Nevertheless, when we repeated the experiment in the presence of ethanol (∼0.65 M), which reacts rapidly (k ) 2 × 109 M-1 s-1) with the hydroxyl radical (15), the intensity of the DMPO/•OH adduct decreased and there was the concomitant appearance of the DMPO/•CH(OH)CH3 adduct (Figure 5C). This finding suggests that at least some of the DMPO/•OH adduct was derived from trapping of the •OH radical. The glutathiyl adduct of DMPO, DMPO/GS•, was detected (Table 2) when berberine and reduced glutathione were irradiated (λ > 300 nm). Irradiation (λ > 390 nm) of an air-saturated acetonitrile solution of berberine and DMPO generated the EPR spectrum shown in Figure 6A. The hfs constants of the major component (92%; aN ) 12.9 G, aH1 ) 10.3 G, aH2 ) 1.2G) identified it as the DMPO/O2•- adduct (16), while the minor component (8%; aN ) 15.0 G, aH ) 15.3) was attributed to the DMPO/•OH adduct (17). When a nitrogen saturated solution of berberine and DMPO in acetonitrile was irradiated (λ > 400 nm) the ESR spectrum shown in Figure 6B was observed. This spectrum consisted of two radicals, one of which was a carbon-centered adduct of DMPO (2, aN ) 15.0 G, aH ) 21.3 G) while the
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Figure 5. (A) EPR spectrum generated by the irradiation (λ > 400 nm) of berberine (4 mM) and DMPO (100 mM) in phosphate buffer (50 mM, pH 7.4). (B) Same as panel A but with SOD (50 µg/mL). (C) Same as panel A but with 0.65 M EtOH.
other consisted of multiple lines with a g-value markedly different from a nitroxide. After further irradiation a second DMPO adduct appeared (3, aN ) 14.2 G, aH ) 18.8 G) (Figure 6C). When the light was switched off the adduct spectra decayed away leaving a multiline spectrum (Figure 6D) which could be simulated using the following parameters: aN) 5.48G, aH ) 7.63 G, 5.45 G, 3.63 G, 3.19 G, and 1.95 G (Figure 6E). A similar but much weaker spectrum was observed when TMPO was used as a spin trap (not shown). No radicals were generated in the absence of DMPO or when PBN was substituted for DMPO. Photoexcited iminium salts, such as berberine, are known to undergo single electrontransfer reactions in the presence of suitable donors (18). Thus, the spectrum in Figure 6D was attributed to the neutral berberine radical (BER•) generated by the one electron reduction of berberine (Figure 1). Table 3 gives
Figure 6. EPR spectrum of berberine (1 mM) and DMPO (100 mM) in acetonitrile irradiated at λ > 400 nm. (A) Air-saturated. (B) Nitrogen sparged. (C) Same as panel B after 15 min. (D) Same as panel C but light off. (E) Simulation using the following hfs parameters for BER• in Table 3. (O) Lines attributable to adduct 2; (X) lines attributable to adduct 3 (see Table 2).
possible assignments of the hfs constants based on calculated spin densities. Adduct 2 was also observed when DMPO alone was irradiated in acetonitrile using the unfiltered arc lamp suggesting that it is derived from the spin trap (for the possible identity of this radical, see ref 19). Adduct 3 may be formed by reaction of DMPO with the BER• radical because its hfs constants are close to the phenyl radical adduct of DMPO (20). MOPAC calculations (Table 3) indicate that the spin density on C-8 of BER• is high enough for DMPO to add to this position. Phototoxicity. Phototoxicity studies were carried out using HaCaT cells, an immortal line of keratinocytes (10).
Table 2. Types of Radicals and the Hyperfine Splitting Constants of Their DMPO Adducts Generated during the UV Irradiation of Berberine in Different Solvents hyperfine coupling constant irradiated compound
solvent
satd. with
berberine
CH3CN
air
berberine
CH3CN
N2
berberine berberine + ethanol (0.65 M) berberine + GSH (1 mM)
phosphate buffer (pH 7.4) phosphate buffer (pH 7.4) phosphate buffer (pH 7.4)
air air air
radical trapped •-
O2 •OH unidentified nitroxide BER• (Figure 1) C• ? (2) C• ? (3) •OH C• ? (1) CH3•CHOH •OH GS• •OH
aN (G)
aH (G)
aH (G)
12.9 15.0 15.2 see Table 3 15.0 14.2 15.2 16.5 16.2 15.2 15.3 15.2
10.3 15.3
1.3
21.3 18.8 14.8 23.3 23.2 15.1 16.2 15.1
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Table 3. Hyperfine Splittings and Spin Densities of Berberine Radical (BER•) position
nucleus
splitting
spin densitya
degeneracy
7 8 11 13 6 12
N H H H H H
5.48 7.63 5.45 3.63 3.19 1.95
(-)0.347 0.533 0.390 0.230 0.028b (-)0.150
1 1 1 1 1 1
a Spin densities were calculated with the Quantum Chemistry Program Exchange program MOPAC, version 6, using the AM1 orbital basis set. b Aliphatic carbon with hydrogen out of plane.
DNA damage increased 2- and 4-fold when the cells were UVA irradiated in the presence of 25 and 50 µM berberine, respectively.
Discussion These studies have shown that the photochemistry of berberine is strongly dependent on its environment. The weak fluorescence of berberine in aqueous solution suggests that either there is rapid intersystem crossing from the singlet to the triplet level or that the molecule returns to the ground state from the singlet state via nonradiative mechanism(s). The latter seems the more plausible explanation as in water no phosphorescence is observed and no 1O2 is generated. In contrast, berberine is fluorescent in dioxane (Table 2), exhibits phosphorescence in ethanol (Figure 2) and generates 1O2 with high quantum yield in CH2Cl2 (Table 2). These observations suggest that berberine photochemistry in the cell will be governed by the subcellular location of the alkaloid. The radical observed during the irradiation of berberine and DMPO in nitrogen sparged acetonitrile, BER•, was identified as the neutral berberine radical (Figure 1). Photoexcited iminium salts, such as berberine, are known to undergo single electron-transfer reactions in the presence of suitable donors such as alcohols (18).
Figure 7. Effect of berberine and UVA (4 J/cm2) on the viability of HaCaT keratinocytes. Berberine concentration (µM) 0 (1); 12.5 (2); 25 (b); 50 (9). SE ) (5%.
Because the BER• radical was not observed in the absence of DMPO, the reducing equivalents may be provided by the spin trap itself,
[BER+]* + DMPO f BER• + DMPO•+
1,3
Figure 8. Comet assay of DNA damage in HaCaT keratinocytes caused by berberine and UVA.
Cells in 96-well dishes were exposed to concentrations of berberine up to 50 µM, then irradiated with increasing doses of UVA and cell viability measured using the MTS assay. No toxicity was observed when cells were exposed to either UVA radiation or berberine alone (Figure 7). However, when UVA irradiated in the presence of berberine there was a dose dependent decrease in cell viability, which fell to 20% at 50 µM berberine and 6J/cm2 UVA. Berberine intercalates into DNA (21), where it undergoes a dramatic increase in fluorescence quantum yield (22). The possibility that the drug could damage DNA was therefore investigated using the Comet assay. As can be seen from Figure 8, DNA damage did not increase when HaCaT keratinocytes were exposed in the dark to concentrations of berberine up to 50 µM; UVA alone at 4J/cm2 caused a small increase in damage. However,
(2)
While the BER• radical was not observed in aqueous solution, the detection of the DMPO/•CH(OH)CH3 and the DMPO/GS• adducts when berberine and DMPO were irradiated in water in the presence of EtOH and GSH, respectively, suggests that a mechanism similar to that shown in eq 1 may also operate in this solvent. The increase in berberine fluorescence when the alkaloid is added to keratinocytes (Figure 3) provides evidence that the alkaloid is present in regions of low polarity. Confocal fluorescence microscopy shows the alkaloid to be present in the mitochondria (Figure 3B), which is in agreement with prior findings (6). The decrease in the viability of HaCaT keratinocytes caused by berberine and UVA (Figure 7) may be the result of membrane damage. Berberine intercalates into DNA (21) where it undergoes a large increase in fluorescence quantum yield (22). DNA damage observed during UVA irradiation of keratinocytes in the presence of berberine (Figure 8) may be due to the generation of one or more of the reactive oxygen species, superoxide, hydroxyl radical, or 1O2, detected in our study. It is also possible that DNA damage occurs via electron transfer from the bases to berberine in a mechanism analogous to that shown in eq 1. A similar mechanism has been proposed for the photosensitization of GG-specific lesions in DNA by UV/ riboflavin (23). However, we were unable to detect any radical products when berberine and deoxyguanosine were irradiated together in aqueous buffer.
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These findings suggest that exposure to sunlight or artificial light sources emitting UVA should be avoided when using topical preparations derived from Goldenseal or containing berberine. However, it is also possible that the efficacy of Goldenseal or berberine as a topical antiseptic is in part due to an interaction between the drug and UVA.
Acknowledgment. The authors thank Dr. A. Motten, NIEHS for performing the MOPAC calculations shown in Table 3 and Dr. D. Miller for recording the confocal microscopy picture shown in Figure 3.
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(11) (12) (13) (14) (15)
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