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Effects of a phytopreparation from Helleborus niger on immunocompetent cells in vitro
Journal of Ethnopharmacology 59 (1998) 139 – 146
Effects of a phytopreparation from Helleborus niger on immunocompetent cells in vitro A. Bu¨ssing a,*, K. Schweizer b a
Krebsforschung Herdecke, Department of Applied Immunology, Communal Hospital Herdecke, Beckweg 4, D-58313 Herdecke, Germany b Institute of Medical Immunology, Technical Uni6ersity (RWTH), Aachen, Germany Received 18 August 1997; received in revised form 5 September 1997; accepted 8 September 1997
Abstract Extracts of Helleborus species are used as phytopreparations with immunostimulatory properties in Romanian traditional medicine. In Germany, Helleborus niger is used in homeopathy and as an adjuvant therapy in the treatment of tumor patients in anthroposophical medicine. In vitro application of an aqueous extract from Helleborus niger resulted in a slight induction of sister chromatid exchanges (SCE) in cultured peripheral blood mononuclear cells (PBMC) from healthy individuals, an effect associated with a slight increase of the [3H]thymidine uptake in the DNA of isolated lymphocytes. Since the cytokines interleukin (IL)-2 and tumor necrosis factor (TNF)-a were reported to increase the number of SCE, we measured the concentrations of these cytokines in the supernatants of cultured PBMC treated with the plant extract. Here, no significant changes were observed as compared with the controls, but a trend to higher supernatant concentrations of TNF-a in six out of ten individuals was noted. Compared with lymphocytes treated with the alkylating substance, cyclophosphamide, the increase of the SCE levels induced by the plant extract is weak. The relevance of this DNA destabilizing property remains to be clarified. © 1998 Elsevier Science Ireland Ltd. Keywords: Helleborus niger; Sister chromatid exchanges; DNA lesions; Immunology; Cytokines
1. Introduction Helleborus species (Ranunculaceae) are used as phytopreparations with immunostimulatory prop* Corresponding author.
erties in Romanian traditional medicine and were reported to possess anti-inflammatory activities in rheumatoid arthritis and varizella zooster infections (Olinescu et al., 1986; Bogdan et al., 1990). To stimulate unspecifically the immune system, roots of Helleborus purpurascens were transcuta-
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neously implanted. In animals treated with this method, an increase of leukocytes, neutrophils and phagocytosis was observed (Bogdan et al., 1990). In Germany, Helleborus niger is used in homeopathy in cases of meningitis, convulsions, hydrocephalus, dropsy and cachexia due to tumors (Metzner, 1985), and it is used as an adjuvant in tumor therapy (subcutaneous injections) in anthroposophical medicine. Among the compounds detected in Helleborus species, there are several steroids such as spirost-5,25(27)-dien-1b,3b,11a-triol (Linde et al., 1971), b-ecdysone (Glombitza et al., 1989), the steroid saponins macranthogenin (Petricic et al., 1969) and macranthoside I (Tschesche et al., 1984), and anemonin, the dilactone of cyclobutane-1,2-diol-1,2-diacrylic acid derived from cyclodimerization of protoanemonin. Recent analytical investigations on Helleborus niger verified the presence of the steroid hormone becdysone, and the steroid saponine macranthosid I (Liedtke et al., in preparation) and confirmed the results of Wissner and Kating (1974), that neither the heart-glycoside helleborin nor other steroidal glycosides were detected. Considering the proposed immunomodulatory and anti-inflammatory properties, and suggesting additional mutagenic effects due to the treatment of patients with cancer and rheumatoid arthritis with this drug, a DNA destabilizing risk has to be defined. To assess possible mutagenic and carcinogenic effects of the drug, we determined the sister chromatid exchange (SCE) frequency of cultured peripheral blood mononuclear cells (PBMC) from healthy individuals after the addition of ‘Helleborus niger D3 aquos’ in vitro. The SCE assay is considered to be a sensitive and rapid method for the detection of agents that damage DNA (Deen et al., 1989; Tucker et al., 1993). SCE represents the interchange of DNA replication products at apparently homologous loci, and involve DNA breakage and reunion of sister chromatids. Thus, we measured SCE of cultured PBMC from healthy individuals treated in vitro with increasing concentrations of an aqueous extract from Helleborus niger.
2. Materials and methods
2.1. Plant material Helleborus niger L. (Ranunculaceae) was collected in O8 tschtal, Austria. The plant was authenticated by the scientific leader of the Botany Garden at the University Marburg, Germany (Prof Dr V. Melzheimer) as Helleborus niger ssp. niger. The voucher samples were deposited at Helixor Herbarium, Rosenfeld, Germany.
2.2. Aqueous extract preparation For our investigations, we used an endotoxinfree aqueous drug extract produced from Helleborus niger. The whole plant material was harvested in summer, while the blossoms were harvested in winter. The plant material was extracted separately by water at 4°C for 2 h (according to HAB 49) and sterilized by filtration. No additional substances like preservatives were added, except sodium chloride for isotonization. Both extracts were mixed 1:1 and diluted three times 1:10 (D3; 3 mg as refered to the fresh plant weight) according to HAB 1. The 1 ml samples of ‘Helleborus niger D3 aquos.’ (Flos rec., Dil. D3 aqueous. 0.5 ml and Plant tota rec., Dil. D3 aqueous. 0.5 ml; 1 ml ampoules; Lot No. C 0211) were kindly provided by Helixor Heilmittel, Rosenfeld (Germany).
2.3. Sister chromatid exchange assay For SCE analysis we used a slight modification of the fluorescence-plus-Giemsa technique that has been described previously (Bu¨ssing et al., 1994). Briefly, heparinized peripheral blood (1 ml) from five healthy male test persons (24–51 years) was cultured in the presence of the thymidine analog 5-bromo-2%-deoxyuridine (BrdU; 5 mg/ml; Sigma) in 9 ml chromosome medium B (Biochrom KG, Berlin, Germany), which contained 2.5 mg/ml phytohemagglutinine (PHA). The drug extract from Helleborus niger was added at final concentrations of 1, 10, 50 or 100 mg/ml. Samples without drug addition served as controls. As a positive control for SCE induc-
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tion, cyclophosphamide (Endoxan; Asta Medica, Frankfurt, Germany) was added at a final concentration of 40 mg/ml. The cells were then cultured in the dark for 70 h at 37°C. Subsequently demecolcine (Colcemid; Biochrome KG) was added and the incubation was continued for additional 2 h. The cells were then suspended in a hypotonic buffer with 0.075 mol/l potassium chloride and fixed on slides with acetic acid/methanol (1:4). The slides were stained with Hoechst 33258 (0.5 mg/ml), exposed to ultraviolet (UV) light (254 nm) for 2 h, incubated for 1 h in 2 ×SSC (sodium chloride–trisodium citrate) buffer at 60°C and counterstained with Giemsa. For scoring SCE, at least 25 M2 metaphases for each sample were recorded. SCE frequency was given in mean SCE number per 46 chromosomes9 S.D. The proliferation index was calculated as described previously (Bu¨ssing et al., 1995a): (1× M1) + (2 × M2) + (3 × M3)/50 metaphases.
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niger was added at final concentrations of 1, 10 or 100 mg/ml. Samples without drug addition served again as controls. Cytokine concentrations in the culture supernatants were determined using sandwitch-ELISA for TNF-a and IL-2 (Quantikine human TNF-a and Quantikine human IL-2; R&D systems, Minneapolis, MN). Cell culture supernatants were stored at −20°C before usage. Samples and standards were run in duplicate in 96-well microtiter plates. Optical density of each well was determined using a spectrophotometer (Dynatech MR 5000) set to 450 nm.
3. Statistical analysis All statistical analysis was done by Wilcoxon’s sign rank test. Differences were considered significant when the probability of the measured differences occurring by chance was B 5%.
2.4. Proliferation assay 4. Results Lymphocyte proliferation was measured by [3H]thymidine incorporation of heparinized peripheral blood lymphocytes, taken from five healthy male individuals (24 – 32 years). Cells were separated by Ficoll-Hypaque sedimentation and cultures were established using 2× 105 cells/ml. ‘Helleborus niger D3 aquos.’ was added at concentrations of 0, 1, 5, 10, 50 or 100 mg/ml, respectively. Controls without added mitogen were used for each culture. After incubation for 96 h at 37°C, cultures were pulsed with 5 mCi/ml [3H]thymidine. After an additional 16 h of culture, harvested cells were washed and scintillation fluid was added. Incorporation of [3H]thymidine of triplicated cultures was determined in counts per minute (cpm) on a LKB 1219 Rackbeta liquid scintillation counter. Proliferation was measured by stimulation index (SI=cpmtest/cpmspontaneous).
2.5. Cytokine assays Heparinized peripheral whole blood (1 ml) from ten healthy male individuals was cultured for 72 h in chromosome medium B (Biochrom KG). The aqueous drug extract from Helleborus
4.1. Sister chromatid exchange assay As shown in Table 1, the mean SCE number of PBMC from five healthy test persons slightly increased by the addition of ‘Helleborus niger D3 aquos’ (P= 0.043). The individual responses revealed a significant increase at a final concentration of 1 mg/ml of the drug in three out of five individuals, while in the other two, the increase was statistically significant at 50 and 100 mg/ml, respectively (Table 1). By the addition of cyclophosphamide, which served as a positive control, 27.99 4.29 SCE/metaphase were observed in cultured PBMC of the whole group. The proliferation index of PBMC treated with the drug (Table 2) showed a slight increase (P= 0.043) at a final concentration of 10 mg/ml of the aqueous drug extract from Helleborus niger, whereas cyclophosphamide decreased the proliferation index (1.69 0.32). Viabilty analysis of cultured cells, measured flow cytometrically by the exclusion of the DNA intercalating dye propidium iodide, revealed no changes as compared with the control samples (data not shown).
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Table 1 Sister chromatid exchanges (SCE) in PBMC SCE/metaphase HN (mg/ml)
9S.D.
0
1
10
50
100
3.87 91.91 5.78 92.78 3.58 91.98 3.03 9 1.99 3.70 9 2.12
4.889 1.93* 6.049 2.12 4.289 2.45 5.209 2.46** 6.099 3.01**
6.05 9 2.49** 6.16 9 2.74 4.06 9 2.08 5.46 9 2.78** 5.479 1.87**
5.51 92.77* 6.38 92.15 5.80 92.45** 5.18 92.67** 5.72 91.96**
5.87 9 2.58** 7.82 9 2.16** 4.42 9 2.06* 5.64 9 2.55* 5.76 9 2.20**
3.99 9 1.05
5.309 0.77***
5.449 0.84***
5.72 90.44***
5.90 9 1.22***
Values represent data of at least 25 M2 metaphases for each healthy test person and are given as means 9S.D. HN (Helleborus niger D3 aquos) was added at 0, 1, 10, 50 or 100 mg/ml cell suspension, respectively. Compared with untreated samples, differences were statistically significant. * P50.03; ** P50.003; *** PB0.05.
4.2. Proliferation assay While the proliferation index more closely reflects the proliferation kinetics of cultured cells, [3H]thymidine is used for labeling newly synthesized DNA in proliferating cells. By measuring the incorporation of [3H]thymidine in the DNA of lymphocytes treated with ‘Helleborus niger D3 aquos’, we observed a slight increase of the [3H]thymidine uptake at 1 mg/ml of the drug in four out of five test persons (Table 3). At higher Table 2 Proliferation index of PBMC Proliferation index HN (mg/ml)
S.D.
0
1
10
50
100
2.25 2.33 1.95 2.33 2.16
2.26 2.20 2.25 2.67 2.57
2.35 2.36 2.32 2.63 2.75
2.29 1.77 2.28 2.67 2.36
2.27 2.46 2.34 2.55 ND
2.20 0.16
2.39 0.21
2.48* 0.20
2.27 0.32
2.43 0.13
Values are given as means 9S.D. of cultured PBMC from healthy male individuals after the addition of HN (Helleborus niger D3 aquos). HN was added at 0, 1, 10, 50 or 100 mg/ml, respectively. * Compared to untreated samples, difference was statistically significant (PB0.05).
concentrations the SI of the whole group decreased, but remained higher than in untreated samples.
4.3. IL-2 and TNF-a in the supernatants of cultured cells The mean IL-2 and TNF-a concentrations in the supernatants of PHA-activated PBMC from ten individuals did not change significantly by the addition of the drug. However, the individual results (Table 4) indicate an individual susceptibility in the responses towards the drug, as in six individuals the TNF-a supernatant concentration increased, but clearly declined in three persons. In unstimulated PBMC, no induction of these cytokines was observed (data not shown).
5. Discussion and conclusions To assess possible mutagenic effects of an aqueous drug extract from Helleborus niger, we investigated the SCE frequency of cultured PBMC from five healthy individuals. While it is obvious that a higher number of tested individuals is obligatory in the exclusion of a possible mutagenic risk of a drug, five experiments with at least 25 M2 metaphases for each tested condition are sufficient to provide strong evidence in this report. We observed a slight induction of SCE at low drug concentrations.
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Table 3 [3H]thymidine incorporation in the DNA of isolated lymphocytes ‘Helleborus niger D3 aquos’ (mg/ml) 0
1
5
10
50
100
cpm
cpm
SI
cpm
SI
cpm
SI
cpm
SI
cpm
SI
cpm
SI
257 252 609 585 279
30 574 24 759 18 633 74 646 35 524
119.0 98.3 30.6 127.6 127.3
60 446 52 324 37 185 58 214 47 628
235.2 207.6 61.1 99.5 170.7
45 227 40 463 41 539 47 319 31 178
176.0 160.6 68.2 80.9 111.7
51 231 47 615 22 659 39 563 25 764
199.3 188.9 37.2 67.6 92.3
66 626 36 296 24 742 53 700 32,318
259.2 144.0 40.6 91.8 115.8
58 650 30 881 18 962 57 039 37,025
228.2 122.5 31.1 97.5 132.7
S.D.
100.5 40.9
154.8 73.0
119.5 47.6
117.1 73.1
130.3 81.5
122.4 71.2
The results of proliferation responses represent the mean values in triplicate cultures from different donors. Values of [3H]thymidine incorporation of PHA-stimulated lymphocytes from five healthy male individuals are expressed in counts per minute (cpm). The stimulation index (SI) was calculated as described in Section 2.
One may argue that alterations of cell proliferation or changes in the subtypes of lymphocytes could account for these effects, as higher SCE levels were found in slowly proliferating cultures than in cultures with a high turn-over rate (Santesson et al., 1979). In addition, T-cell cultures showed a higher SCE frequency but a slower rate of turn-over than B-cells (Santesson et al., 1979; Lindblad and Lambert, 1981). However, in our experiments, the proliferation index slightly increased, whereas the amount of CD3 + T-cells remained almost unchanged (data not shown). Using isolated lymphocytes, the [3H]thymidine incorporation in the DNA of lymphocytes treated with the drug slightly increased. Obviously, these explanations cannot be attributed to the observed SCE induction. Since an induction of SCE by cytokines such as IL-2 and TNF-a was reported previously (Morris et al., 1990; Lazutka and Rudaitiene, 1992), one may suggest that TNF-a, slightly elevated in ‘Helleborus niger D3 aquos’treated samples in six out of ten individuals, might contribute to the observed SCE induction. In lymphocytes treated with Viscum album L. (Loranthaceae) or purified mistletoe lectins, also an induction of TNF-a was observed (Hajto et al., 1990), but the SCE decreased by the addition of a plant extract from Viscum album L. (Bu¨ssing et
al., 1994, 1995b, 1996). Thus, it seems unlikely that the slight release of TNF-a is the main causative mechanism for the observed slight induction of SCE. In PBMC treated with the streptococcal pyrogenic exotoxins A and C (Bu¨ssing et al., 1995d), both known as superantigens with proliferationinducing properties, and also in PBMC treated with the Helleborus niger-extract, we observed a slight increase in the uptake of [3H]thymidine in the lymphocytes and a slight property of DNA destabilization, while in PBMC treated with a drug extract from Viscum album L., both, the SCE and the uptake of [3H]thymidine decreased (Bu¨ssing et al., 1994, 1995c). Thus, there is an obvious association between the drug-mediated uptake of [3H]thymidine and DNA-destabilization. However, on the T-cell surface the expression of activation-associated molecules such as interleukin-2 receptor a chains (CD25) and transferrin receptors (CD71), which both are prerequisites for cell proliferation (Neckers and Cossman, 1983), did not change by the addition of the Helleborus niger-extract (data not shown), indicating that the drug itself did not induce blast cell transformation. The level of SCE observed in response to the drug is completely different to that observed in
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Table 4 Concentration of TNF-a and IL-2 in the supernatants of PHA-activated PBMC TNF-a (pg/ml) HN (mg/ml)
SD
IL-2 (pg/ml)
0
1
10
100
0
1
10
100
636.8 779.5 823.1 792.2 1427.9 1240.8 904.1 1026.5 520.1 1841.5
563.9 861.3 997.7 827.2 1617.5 1394.3 992.8 1448.4 439.8 1441.5
567.5 857.0 955.4 1017.7 1635.9 1367.7 1054.5 1503.9 294.6 1591.4
420.8 981.3 1030.5 934.0 1203.3 1673.3 976.5 1540.1 305.1 1554.7
76.8 266.7 132.7 246.8 243.5 216.5 197.1 57.5 82.2 119.0
47.0 298.5 156.6 254.3 271.2 228.3 228.3 56.3 58.4 117.3
58.7 258.2 150.7 262.6 229.4 265.6 231.2 48.0 54.6 105.6
44.3 252.4 114.2 247.3 112.7 313.8 249.5 ND 49.7 69.1
993.3 400.6
1058.4 401.6
1084.6 445.0
1062.0 457.1
163.9 79.0
171.6 96.7
166.5 93.0
176.7 109.0
All results are shown as the mean values from duplicated samples of ten different donors. HN (Helleborus niger D3 aquos) was added at 0, 1, 10 or 100 mg/ml, respectively.
cyclophosphamide-treated samples. A severe increase of SCE-inducing DNA lesions by antirheumatic agents such as cyclophosphamide and cyclosporine is a well known effect (Yuzawa et al., 1986; Wilmer et al., 1992). Although SCE-inducing DNA lesions may be of crucial importance for mutagenesis and the development of secondary neoplasms (Lambert et al., 1978), these lesions are in generally not permanent (Raposa, 1978). As reported previously, T-lymphocytes with very high cyclophosphamide-induced SCE will die within 1 week, whereas less damaged cells seem to survive, however, with higher SCE levels (Mertens et al., 1995); their fate is unclear. Although cyclosporine was found to induce SCE in vitro, long-term treatment of heart transplant recipients with cyclosporine did not alter their SCE in vivo (Bu¨ssing et al., unpublished observations). Thus, depending on administered drug concentrations, the observed effects might not be relevant in vivo. Boicil, a complex extract of different Helleborus species, was reported to inhibit mitogenesis of PHA-stimulated lymphocytes (Olinescu et al., 1986). Using ‘Helleborus niger D3 aquos’ in our experiments, the [3H]thymidine incorporation in the DNA of isolated lymphocytes slightly increased. These contradictory results might be due
to different drug components present in Helleborus species. As it was reported by Dirsch et al. (1993), hellebrin, b-ecdysone and 5a-hydroxyecdysone from Helleborus purpurascens suppressed lymphocyte proliferation, whereas an isolated steroidal saponin stimulated the proliferation. However, neither hellebrin nor other steroidal glycosides but some undefined saponins were detected in Helleborus niger (Wissner and Kating, 1974). In the material used for our experiments, b-ecdysone, macranthosid I, protoanemonin and flavonoids were detected (E. Lorch, personal communication). Although at present uncharacterized, a component with some agglutinin activity was found too. However, identification and characterization of components involved in the induction of the observed effects will be of prime importance in elucidating the underlying mechanisms. At present, there are no investigations available on the DNA-affecting properties of the various compounds from Helleborus species. In conclusion, our results indicate that the aqueous drug extract from Helleborus niger possesses immunomodulating properties but also exerts slight effects of DNA destabilization. These effects showed interindividual differences. Al-
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though the significance of the observed slight SCE induction by ‘Helleborus niger D3 aquos’ remaind speculative, the data suggest that the drug may have a mutagenic effect on human PBMC. These preliminary results should encourage further studies of the properties of this drug. At present, we try to define the main active components, and whether the in vitro results are of relevance in vivo.
Acknowledgements The technical assistance of Ilse Seyfarth-Uhlemann and Kiliana Suzart is gratefully acknowledged. We are also grateful to Helixor Heilmittel, Rosenfeld (Germany) for kindly providing the ‘Helleborus niger D3 aquos’ samples. Thanks to Dr E. Lorch for sharing unpublished results.
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Effects of a phytopreparation from Helleborus niger on immunocompetent cells in vitro A. Bu¨ssing a,*, K. Schweizer b a
Krebsforschung Herdecke, Department of Applied Immunology, Communal Hospital Herdecke, Beckweg 4, D-58313 Herdecke, Germany b Institute of Medical Immunology, Technical Uni6ersity (RWTH), Aachen, Germany Received 18 August 1997; received in revised form 5 September 1997; accepted 8 September 1997
Abstract Extracts of Helleborus species are used as phytopreparations with immunostimulatory properties in Romanian traditional medicine. In Germany, Helleborus niger is used in homeopathy and as an adjuvant therapy in the treatment of tumor patients in anthroposophical medicine. In vitro application of an aqueous extract from Helleborus niger resulted in a slight induction of sister chromatid exchanges (SCE) in cultured peripheral blood mononuclear cells (PBMC) from healthy individuals, an effect associated with a slight increase of the [3H]thymidine uptake in the DNA of isolated lymphocytes. Since the cytokines interleukin (IL)-2 and tumor necrosis factor (TNF)-a were reported to increase the number of SCE, we measured the concentrations of these cytokines in the supernatants of cultured PBMC treated with the plant extract. Here, no significant changes were observed as compared with the controls, but a trend to higher supernatant concentrations of TNF-a in six out of ten individuals was noted. Compared with lymphocytes treated with the alkylating substance, cyclophosphamide, the increase of the SCE levels induced by the plant extract is weak. The relevance of this DNA destabilizing property remains to be clarified. © 1998 Elsevier Science Ireland Ltd. Keywords: Helleborus niger; Sister chromatid exchanges; DNA lesions; Immunology; Cytokines
1. Introduction Helleborus species (Ranunculaceae) are used as phytopreparations with immunostimulatory prop* Corresponding author.
erties in Romanian traditional medicine and were reported to possess anti-inflammatory activities in rheumatoid arthritis and varizella zooster infections (Olinescu et al., 1986; Bogdan et al., 1990). To stimulate unspecifically the immune system, roots of Helleborus purpurascens were transcuta-
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neously implanted. In animals treated with this method, an increase of leukocytes, neutrophils and phagocytosis was observed (Bogdan et al., 1990). In Germany, Helleborus niger is used in homeopathy in cases of meningitis, convulsions, hydrocephalus, dropsy and cachexia due to tumors (Metzner, 1985), and it is used as an adjuvant in tumor therapy (subcutaneous injections) in anthroposophical medicine. Among the compounds detected in Helleborus species, there are several steroids such as spirost-5,25(27)-dien-1b,3b,11a-triol (Linde et al., 1971), b-ecdysone (Glombitza et al., 1989), the steroid saponins macranthogenin (Petricic et al., 1969) and macranthoside I (Tschesche et al., 1984), and anemonin, the dilactone of cyclobutane-1,2-diol-1,2-diacrylic acid derived from cyclodimerization of protoanemonin. Recent analytical investigations on Helleborus niger verified the presence of the steroid hormone becdysone, and the steroid saponine macranthosid I (Liedtke et al., in preparation) and confirmed the results of Wissner and Kating (1974), that neither the heart-glycoside helleborin nor other steroidal glycosides were detected. Considering the proposed immunomodulatory and anti-inflammatory properties, and suggesting additional mutagenic effects due to the treatment of patients with cancer and rheumatoid arthritis with this drug, a DNA destabilizing risk has to be defined. To assess possible mutagenic and carcinogenic effects of the drug, we determined the sister chromatid exchange (SCE) frequency of cultured peripheral blood mononuclear cells (PBMC) from healthy individuals after the addition of ‘Helleborus niger D3 aquos’ in vitro. The SCE assay is considered to be a sensitive and rapid method for the detection of agents that damage DNA (Deen et al., 1989; Tucker et al., 1993). SCE represents the interchange of DNA replication products at apparently homologous loci, and involve DNA breakage and reunion of sister chromatids. Thus, we measured SCE of cultured PBMC from healthy individuals treated in vitro with increasing concentrations of an aqueous extract from Helleborus niger.
2. Materials and methods
2.1. Plant material Helleborus niger L. (Ranunculaceae) was collected in O8 tschtal, Austria. The plant was authenticated by the scientific leader of the Botany Garden at the University Marburg, Germany (Prof Dr V. Melzheimer) as Helleborus niger ssp. niger. The voucher samples were deposited at Helixor Herbarium, Rosenfeld, Germany.
2.2. Aqueous extract preparation For our investigations, we used an endotoxinfree aqueous drug extract produced from Helleborus niger. The whole plant material was harvested in summer, while the blossoms were harvested in winter. The plant material was extracted separately by water at 4°C for 2 h (according to HAB 49) and sterilized by filtration. No additional substances like preservatives were added, except sodium chloride for isotonization. Both extracts were mixed 1:1 and diluted three times 1:10 (D3; 3 mg as refered to the fresh plant weight) according to HAB 1. The 1 ml samples of ‘Helleborus niger D3 aquos.’ (Flos rec., Dil. D3 aqueous. 0.5 ml and Plant tota rec., Dil. D3 aqueous. 0.5 ml; 1 ml ampoules; Lot No. C 0211) were kindly provided by Helixor Heilmittel, Rosenfeld (Germany).
2.3. Sister chromatid exchange assay For SCE analysis we used a slight modification of the fluorescence-plus-Giemsa technique that has been described previously (Bu¨ssing et al., 1994). Briefly, heparinized peripheral blood (1 ml) from five healthy male test persons (24–51 years) was cultured in the presence of the thymidine analog 5-bromo-2%-deoxyuridine (BrdU; 5 mg/ml; Sigma) in 9 ml chromosome medium B (Biochrom KG, Berlin, Germany), which contained 2.5 mg/ml phytohemagglutinine (PHA). The drug extract from Helleborus niger was added at final concentrations of 1, 10, 50 or 100 mg/ml. Samples without drug addition served as controls. As a positive control for SCE induc-
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tion, cyclophosphamide (Endoxan; Asta Medica, Frankfurt, Germany) was added at a final concentration of 40 mg/ml. The cells were then cultured in the dark for 70 h at 37°C. Subsequently demecolcine (Colcemid; Biochrome KG) was added and the incubation was continued for additional 2 h. The cells were then suspended in a hypotonic buffer with 0.075 mol/l potassium chloride and fixed on slides with acetic acid/methanol (1:4). The slides were stained with Hoechst 33258 (0.5 mg/ml), exposed to ultraviolet (UV) light (254 nm) for 2 h, incubated for 1 h in 2 ×SSC (sodium chloride–trisodium citrate) buffer at 60°C and counterstained with Giemsa. For scoring SCE, at least 25 M2 metaphases for each sample were recorded. SCE frequency was given in mean SCE number per 46 chromosomes9 S.D. The proliferation index was calculated as described previously (Bu¨ssing et al., 1995a): (1× M1) + (2 × M2) + (3 × M3)/50 metaphases.
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niger was added at final concentrations of 1, 10 or 100 mg/ml. Samples without drug addition served again as controls. Cytokine concentrations in the culture supernatants were determined using sandwitch-ELISA for TNF-a and IL-2 (Quantikine human TNF-a and Quantikine human IL-2; R&D systems, Minneapolis, MN). Cell culture supernatants were stored at −20°C before usage. Samples and standards were run in duplicate in 96-well microtiter plates. Optical density of each well was determined using a spectrophotometer (Dynatech MR 5000) set to 450 nm.
3. Statistical analysis All statistical analysis was done by Wilcoxon’s sign rank test. Differences were considered significant when the probability of the measured differences occurring by chance was B 5%.
2.4. Proliferation assay 4. Results Lymphocyte proliferation was measured by [3H]thymidine incorporation of heparinized peripheral blood lymphocytes, taken from five healthy male individuals (24 – 32 years). Cells were separated by Ficoll-Hypaque sedimentation and cultures were established using 2× 105 cells/ml. ‘Helleborus niger D3 aquos.’ was added at concentrations of 0, 1, 5, 10, 50 or 100 mg/ml, respectively. Controls without added mitogen were used for each culture. After incubation for 96 h at 37°C, cultures were pulsed with 5 mCi/ml [3H]thymidine. After an additional 16 h of culture, harvested cells were washed and scintillation fluid was added. Incorporation of [3H]thymidine of triplicated cultures was determined in counts per minute (cpm) on a LKB 1219 Rackbeta liquid scintillation counter. Proliferation was measured by stimulation index (SI=cpmtest/cpmspontaneous).
2.5. Cytokine assays Heparinized peripheral whole blood (1 ml) from ten healthy male individuals was cultured for 72 h in chromosome medium B (Biochrom KG). The aqueous drug extract from Helleborus
4.1. Sister chromatid exchange assay As shown in Table 1, the mean SCE number of PBMC from five healthy test persons slightly increased by the addition of ‘Helleborus niger D3 aquos’ (P= 0.043). The individual responses revealed a significant increase at a final concentration of 1 mg/ml of the drug in three out of five individuals, while in the other two, the increase was statistically significant at 50 and 100 mg/ml, respectively (Table 1). By the addition of cyclophosphamide, which served as a positive control, 27.99 4.29 SCE/metaphase were observed in cultured PBMC of the whole group. The proliferation index of PBMC treated with the drug (Table 2) showed a slight increase (P= 0.043) at a final concentration of 10 mg/ml of the aqueous drug extract from Helleborus niger, whereas cyclophosphamide decreased the proliferation index (1.69 0.32). Viabilty analysis of cultured cells, measured flow cytometrically by the exclusion of the DNA intercalating dye propidium iodide, revealed no changes as compared with the control samples (data not shown).
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Table 1 Sister chromatid exchanges (SCE) in PBMC SCE/metaphase HN (mg/ml)
9S.D.
0
1
10
50
100
3.87 91.91 5.78 92.78 3.58 91.98 3.03 9 1.99 3.70 9 2.12
4.889 1.93* 6.049 2.12 4.289 2.45 5.209 2.46** 6.099 3.01**
6.05 9 2.49** 6.16 9 2.74 4.06 9 2.08 5.46 9 2.78** 5.479 1.87**
5.51 92.77* 6.38 92.15 5.80 92.45** 5.18 92.67** 5.72 91.96**
5.87 9 2.58** 7.82 9 2.16** 4.42 9 2.06* 5.64 9 2.55* 5.76 9 2.20**
3.99 9 1.05
5.309 0.77***
5.449 0.84***
5.72 90.44***
5.90 9 1.22***
Values represent data of at least 25 M2 metaphases for each healthy test person and are given as means 9S.D. HN (Helleborus niger D3 aquos) was added at 0, 1, 10, 50 or 100 mg/ml cell suspension, respectively. Compared with untreated samples, differences were statistically significant. * P50.03; ** P50.003; *** PB0.05.
4.2. Proliferation assay While the proliferation index more closely reflects the proliferation kinetics of cultured cells, [3H]thymidine is used for labeling newly synthesized DNA in proliferating cells. By measuring the incorporation of [3H]thymidine in the DNA of lymphocytes treated with ‘Helleborus niger D3 aquos’, we observed a slight increase of the [3H]thymidine uptake at 1 mg/ml of the drug in four out of five test persons (Table 3). At higher Table 2 Proliferation index of PBMC Proliferation index HN (mg/ml)
S.D.
0
1
10
50
100
2.25 2.33 1.95 2.33 2.16
2.26 2.20 2.25 2.67 2.57
2.35 2.36 2.32 2.63 2.75
2.29 1.77 2.28 2.67 2.36
2.27 2.46 2.34 2.55 ND
2.20 0.16
2.39 0.21
2.48* 0.20
2.27 0.32
2.43 0.13
Values are given as means 9S.D. of cultured PBMC from healthy male individuals after the addition of HN (Helleborus niger D3 aquos). HN was added at 0, 1, 10, 50 or 100 mg/ml, respectively. * Compared to untreated samples, difference was statistically significant (PB0.05).
concentrations the SI of the whole group decreased, but remained higher than in untreated samples.
4.3. IL-2 and TNF-a in the supernatants of cultured cells The mean IL-2 and TNF-a concentrations in the supernatants of PHA-activated PBMC from ten individuals did not change significantly by the addition of the drug. However, the individual results (Table 4) indicate an individual susceptibility in the responses towards the drug, as in six individuals the TNF-a supernatant concentration increased, but clearly declined in three persons. In unstimulated PBMC, no induction of these cytokines was observed (data not shown).
5. Discussion and conclusions To assess possible mutagenic effects of an aqueous drug extract from Helleborus niger, we investigated the SCE frequency of cultured PBMC from five healthy individuals. While it is obvious that a higher number of tested individuals is obligatory in the exclusion of a possible mutagenic risk of a drug, five experiments with at least 25 M2 metaphases for each tested condition are sufficient to provide strong evidence in this report. We observed a slight induction of SCE at low drug concentrations.
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Table 3 [3H]thymidine incorporation in the DNA of isolated lymphocytes ‘Helleborus niger D3 aquos’ (mg/ml) 0
1
5
10
50
100
cpm
cpm
SI
cpm
SI
cpm
SI
cpm
SI
cpm
SI
cpm
SI
257 252 609 585 279
30 574 24 759 18 633 74 646 35 524
119.0 98.3 30.6 127.6 127.3
60 446 52 324 37 185 58 214 47 628
235.2 207.6 61.1 99.5 170.7
45 227 40 463 41 539 47 319 31 178
176.0 160.6 68.2 80.9 111.7
51 231 47 615 22 659 39 563 25 764
199.3 188.9 37.2 67.6 92.3
66 626 36 296 24 742 53 700 32,318
259.2 144.0 40.6 91.8 115.8
58 650 30 881 18 962 57 039 37,025
228.2 122.5 31.1 97.5 132.7
S.D.
100.5 40.9
154.8 73.0
119.5 47.6
117.1 73.1
130.3 81.5
122.4 71.2
The results of proliferation responses represent the mean values in triplicate cultures from different donors. Values of [3H]thymidine incorporation of PHA-stimulated lymphocytes from five healthy male individuals are expressed in counts per minute (cpm). The stimulation index (SI) was calculated as described in Section 2.
One may argue that alterations of cell proliferation or changes in the subtypes of lymphocytes could account for these effects, as higher SCE levels were found in slowly proliferating cultures than in cultures with a high turn-over rate (Santesson et al., 1979). In addition, T-cell cultures showed a higher SCE frequency but a slower rate of turn-over than B-cells (Santesson et al., 1979; Lindblad and Lambert, 1981). However, in our experiments, the proliferation index slightly increased, whereas the amount of CD3 + T-cells remained almost unchanged (data not shown). Using isolated lymphocytes, the [3H]thymidine incorporation in the DNA of lymphocytes treated with the drug slightly increased. Obviously, these explanations cannot be attributed to the observed SCE induction. Since an induction of SCE by cytokines such as IL-2 and TNF-a was reported previously (Morris et al., 1990; Lazutka and Rudaitiene, 1992), one may suggest that TNF-a, slightly elevated in ‘Helleborus niger D3 aquos’treated samples in six out of ten individuals, might contribute to the observed SCE induction. In lymphocytes treated with Viscum album L. (Loranthaceae) or purified mistletoe lectins, also an induction of TNF-a was observed (Hajto et al., 1990), but the SCE decreased by the addition of a plant extract from Viscum album L. (Bu¨ssing et
al., 1994, 1995b, 1996). Thus, it seems unlikely that the slight release of TNF-a is the main causative mechanism for the observed slight induction of SCE. In PBMC treated with the streptococcal pyrogenic exotoxins A and C (Bu¨ssing et al., 1995d), both known as superantigens with proliferationinducing properties, and also in PBMC treated with the Helleborus niger-extract, we observed a slight increase in the uptake of [3H]thymidine in the lymphocytes and a slight property of DNA destabilization, while in PBMC treated with a drug extract from Viscum album L., both, the SCE and the uptake of [3H]thymidine decreased (Bu¨ssing et al., 1994, 1995c). Thus, there is an obvious association between the drug-mediated uptake of [3H]thymidine and DNA-destabilization. However, on the T-cell surface the expression of activation-associated molecules such as interleukin-2 receptor a chains (CD25) and transferrin receptors (CD71), which both are prerequisites for cell proliferation (Neckers and Cossman, 1983), did not change by the addition of the Helleborus niger-extract (data not shown), indicating that the drug itself did not induce blast cell transformation. The level of SCE observed in response to the drug is completely different to that observed in
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Table 4 Concentration of TNF-a and IL-2 in the supernatants of PHA-activated PBMC TNF-a (pg/ml) HN (mg/ml)
SD
IL-2 (pg/ml)
0
1
10
100
0
1
10
100
636.8 779.5 823.1 792.2 1427.9 1240.8 904.1 1026.5 520.1 1841.5
563.9 861.3 997.7 827.2 1617.5 1394.3 992.8 1448.4 439.8 1441.5
567.5 857.0 955.4 1017.7 1635.9 1367.7 1054.5 1503.9 294.6 1591.4
420.8 981.3 1030.5 934.0 1203.3 1673.3 976.5 1540.1 305.1 1554.7
76.8 266.7 132.7 246.8 243.5 216.5 197.1 57.5 82.2 119.0
47.0 298.5 156.6 254.3 271.2 228.3 228.3 56.3 58.4 117.3
58.7 258.2 150.7 262.6 229.4 265.6 231.2 48.0 54.6 105.6
44.3 252.4 114.2 247.3 112.7 313.8 249.5 ND 49.7 69.1
993.3 400.6
1058.4 401.6
1084.6 445.0
1062.0 457.1
163.9 79.0
171.6 96.7
166.5 93.0
176.7 109.0
All results are shown as the mean values from duplicated samples of ten different donors. HN (Helleborus niger D3 aquos) was added at 0, 1, 10 or 100 mg/ml, respectively.
cyclophosphamide-treated samples. A severe increase of SCE-inducing DNA lesions by antirheumatic agents such as cyclophosphamide and cyclosporine is a well known effect (Yuzawa et al., 1986; Wilmer et al., 1992). Although SCE-inducing DNA lesions may be of crucial importance for mutagenesis and the development of secondary neoplasms (Lambert et al., 1978), these lesions are in generally not permanent (Raposa, 1978). As reported previously, T-lymphocytes with very high cyclophosphamide-induced SCE will die within 1 week, whereas less damaged cells seem to survive, however, with higher SCE levels (Mertens et al., 1995); their fate is unclear. Although cyclosporine was found to induce SCE in vitro, long-term treatment of heart transplant recipients with cyclosporine did not alter their SCE in vivo (Bu¨ssing et al., unpublished observations). Thus, depending on administered drug concentrations, the observed effects might not be relevant in vivo. Boicil, a complex extract of different Helleborus species, was reported to inhibit mitogenesis of PHA-stimulated lymphocytes (Olinescu et al., 1986). Using ‘Helleborus niger D3 aquos’ in our experiments, the [3H]thymidine incorporation in the DNA of isolated lymphocytes slightly increased. These contradictory results might be due
to different drug components present in Helleborus species. As it was reported by Dirsch et al. (1993), hellebrin, b-ecdysone and 5a-hydroxyecdysone from Helleborus purpurascens suppressed lymphocyte proliferation, whereas an isolated steroidal saponin stimulated the proliferation. However, neither hellebrin nor other steroidal glycosides but some undefined saponins were detected in Helleborus niger (Wissner and Kating, 1974). In the material used for our experiments, b-ecdysone, macranthosid I, protoanemonin and flavonoids were detected (E. Lorch, personal communication). Although at present uncharacterized, a component with some agglutinin activity was found too. However, identification and characterization of components involved in the induction of the observed effects will be of prime importance in elucidating the underlying mechanisms. At present, there are no investigations available on the DNA-affecting properties of the various compounds from Helleborus species. In conclusion, our results indicate that the aqueous drug extract from Helleborus niger possesses immunomodulating properties but also exerts slight effects of DNA destabilization. These effects showed interindividual differences. Al-
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though the significance of the observed slight SCE induction by ‘Helleborus niger D3 aquos’ remaind speculative, the data suggest that the drug may have a mutagenic effect on human PBMC. These preliminary results should encourage further studies of the properties of this drug. At present, we try to define the main active components, and whether the in vitro results are of relevance in vivo.
Acknowledgements The technical assistance of Ilse Seyfarth-Uhlemann and Kiliana Suzart is gratefully acknowledged. We are also grateful to Helixor Heilmittel, Rosenfeld (Germany) for kindly providing the ‘Helleborus niger D3 aquos’ samples. Thanks to Dr E. Lorch for sharing unpublished results.
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