J Biol Inorg Chem DOI 10.1007/s00775-014-1135-4
ORIGINAL PAPER
Historical and biochemical aspects of a seventeenth century gold-based aurum vitae recipe Riccardo Rubbiani • Bettina Wahrig Ingo Ott
•
Received: 1 December 2013 / Accepted: 29 March 2014 Ó SBIC 2014
Abstract The medicinal chemistry and biomedical applications of gold complexes have been intensively studied over the last decades. Some complexes have been used for the treatment of rheumatoid arthritis, and a considerable number of new metallodrug candidates have been developed as new anticancer drugs and anti-infectives. However, the therapeutic use of gold and its complexes goes back to ancient times and was also of great importance for alchemists until the modern age. In this report, we give an overview of the alchemic medicine between the sixteenth and the early eighteenth century and describe the cytotoxicity and thioredoxin reductase (TrxR) inhibition of a typical ‘‘aurum vitae’’ medicine, which was prepared according to a recipe by Bartholoma¨us Kretschmar from the seventeenth century. ‘‘Aurum vitae’’ consists of a mixture of gold, mercury and antimony complexes and shows the expected cytotoxic and TrxR inhibitory properties providing some rationale for therapeutic effects of this kind of historical medicinal preparation.
Electronic supplementary material The online version of this article (doi:10.1007/s00775-014-1135-4) contains supplementary material, which is available to authorized users. R. Rubbiani I. Ott (&) Institute of Medicinal and Pharmaceutical Chemistry, Technische Universita¨t Braunschweig, Beethovenstraße 55, 38106 Braunschweig, Germany e-mail: [email protected] B. Wahrig Pharmazie- und Wissenschaftsgeschichte, Technische Universita¨t Braunschweig, Beethovenstraße 55, 38106 Braunschweig, Germany
Keywords Alchemy Antimony Aurum vitae Cytotoxicity Gold
Introduction The last decades have witnessed a steadily increasing interest in the biochemistry and biomedical applications of gold complexes [1]. Gold-based drugs such as Ridaura (auranofin), Myocrisin (aurothiomalate) and Solgonal (aurothioglucose) are nowadays used in the treatment of rheumatoid arthritis [2]. Moreover, gold(I) and gold(III) complexes have been demonstrating promising anticancer, antiviral and antiparasitic properties in many preclinical studies [3–7]. However, while most of the current understanding of the biomedical properties of gold species was established during the last century [8–10], gold preparations were already involved in the trades and in medicine more than 4,000 years ago [11, 12]. Gold-based remedies have been believed to be effective against a considerable number of diseases since ancient times and were also produced by many alchemists of medieval until modern ages [13–19]. Alchemy declined with the development of modern science, but many experimental and basic laboratory procedures are still recognizable today. However, historical preparations are not widely accessible today and the descriptions how to prepare those are related to pharmaceutical and biomedical concepts of the respective time. Accordingly, a reinvestigation of historical gold remedies appears not to have been performed so far. With this in mind, we report in this paper on historical aspects of medical gold preparations between the sixteenth and twentieth century and on selected biochemical key properties of a seventeenth century gold-based medicine, which we have prepared according to a historical procedure.
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Materials and methods General All solvents were used as received from Sigma-Aldrich or Fluka. Gold powder was purchased from ChemPur, mercury was purchased from Acros Organic, antimony(III) sulfide was purchased from Sigma-Aldrich.
concentration over time followed a linear trend (r2 C 0.99), and the enzymatic activities were calculated as the slopes (increase in absorbance per second) thereof. For each tested compound, the non-interference with the assay components was confirmed by a negative control experiment using an enzyme-free solution. The IC50 values were calculated as the concentration of compound decreasing the enzymatic activity of the untreated control by 50 % and are given as the means and errors of 3–6 independent experiments.
Aurum vitae preparation Antiproliferative experiments 0.150 g of gold (0.38 mmol) was dissolved in 5–10 mL of ‘‘aqua regis’’ (conc HNO3:conc HCl, 1:3) forming solution A. 0.750 g of mercury (1.87 mmol) was dissolved in HNO3 50 % giving solution B. 0.150 g of antimony sulfide Sb2S3 (0.44 mmol) was dissolved in ‘‘aqua regis’’ (conc HNO3:conc HCl, 1:3) forming solution C. Solution A, B and C were mixed and heated under reflux conditions and vigorous stirring for 12 h. The suspension was allowed to cool down and treated with 20 mL of ethanol absolute and 20 mL of distilled H2O. The obtained solution was distilled at 100 °C for 6 h and the distillate was discarded. A volume of 50 mL of ethanol 96 % was added, the solution was distilled at 100 °C and the distillate discarded. The last step was repeated 6 times. The finally obtained residue was placed in muffle furnace at 600 °C for 24 h and turned into a yellow–brown solid. Enzyme inhibition assay To determine the inhibition of TrxR and GR, an established microplate reader-based assay was performed [20]. For this purpose, commercially available rat liver TrxR or baker yeast GR (both from Sigma-Aldrich) were used and diluted with distilled water to achieve a concentration of 2.0 U/mL. The compounds were freshly dissolved as stock solutions in DMF. To each 25 lL aliquot of the enzyme solution, each 25 lL of potassium phosphate buffer pH 7.0 containing the compounds in graded concentrations or vehicle (DMF) without compounds (control probe) was added, and the resulting solutions (final concentration of DMF: max. 0.5 % v/v) were incubated with moderate shaking for 75 min at 37 °C in a 96-well plate. To each well 225 lL of reaction mixture (1,000 lL of reaction mixture consisted of 500 lL of potassium phosphate buffer, pH 7.0, 80 lL of 100 mM EDTA solution, pH 7.5, 20 lL of 0.05 % BSA solution, 100 lL of 20 mM NADPH solution, and 300 lL of distilled) water were added, and the reaction was started by the addition of 25 lL of an 20 mM ethanolic 5,50 -dithio-bis-nitrobenzoic acid (DTNB) solution. After proper mixing, the formation of 5-thio-nitrobenzoic acid (5-TNB) was monitored with a microplate reader (Perkin-Elmer VictorX4) at 405 nm in 10-s intervals for 6 min. The increase in 5-TNB
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The antiproliferative effects in MCF-7 and HT-29 cells after 72 h (HT-29) or 96 h (MCF-7) exposure to the gold preparations were evaluated according to a procedure already described in the literature [20]. For the experiments, the compounds were prepared freshly as stock solutions in DMF and diluted with the cell culture medium to the final assay concentrations (0.1 % v/v DMF). The cells were cultured in a 75 cm2 flask with 10 mL of cell culture medium. A volume of 100 lL of 12,000 cells/mL medium (MCF-7) or 6,000 cells/mL (HT-29) was seeded in 96-well plates and incubated for 48 (HT-29) or 72 h (MCF-7) at 37 °C under 5 % CO2. Afterward, the medium was removed and replaced with medium containing the substances in different concentrations. The cells were exposed to the complexes for 72 (HT-29) or 96 h (MCF-7). The medium was removed, and the cells were washed with PBS and stained with 100 lL of 0.02 % crystal violet for 30 min. The excess of crystal violet was removed, the plates were washed with water and dried, 180 lL of 70 % ethanol was added to each well, and the plates were read at 595 nm in a microplate reader (PerkinElmer VictorX4) after 3–4 h of gentle shaking. The IC50 values were calculated as the concentrations reducing proliferation of untreated control cells by 50 % and are given as the means and errors of at least two independent experiments (each performed with n = 6).
Results and discussion Overview of the alchemic medicine between the sixteenth and the early eighteenth century Alchemic preparations started with crude drugs, and they included often a large number of elaborate and time-consuming operations. These were considered to separate the beneficial properties from the toxic effects of a substance developing a kind of therapeutic essence. Gold played a key role within the alchemic tradition [21]. The deep interest of the alchemists in this element was related to their efforts to undertake material transformations of one element into the others with the final aim to transmute base
J Biol Inorg Chem
metals into gold. In this context, the ‘‘lapis philosophorum’’ or philosopher’s stone was a mediating substance, which many alchemists sought to produce [22]. But although the philosopher’s stone was probably never created, the growing (al)chemical knowledge of specific procedures found many applications. Alchemy became a field of applied knowledge, may be even of ‘‘technoscience avant la lettre’’ [23]. Besides their close relationship to mining and smelting, glass and pottery production, alchemical substances found many medicinal applications [24]. Among them, gold-based remedies were often considered a sort of ‘‘panacea’’, a remedy for all or nearly all the diseases. Moreover, gold medicaments were thought to have rejuvenating properties because of the glittering yellow color and the comparison with the divinized ‘‘Sun’’ [11, 12, 21]. Before the alchemical era, gold was administered as solid gold leaves suspended in wine, ‘‘Spiritus’’ (distilled alcohol) and fruit or plants juice and this tradition was continued in official pharmacopeas and within pharmaceutical traditions outside alchemy [12, 25]. However, the alchemists came to the conclusion that a liquid gold preparation could have more chances to address a disease, because it would dissipate the noxious humors which stood at the origin of it. To achieve this status, the transformation of gold was essential. Philippus Theophrastus Aureolus Bombastus von Hohenheim (also called Paracelsus) elaborated both the concept of ‘‘arcanum’’ and gold preparations, among them ‘‘aurum potabile’’ which most likely existed already before him [26]. According to the theory of ‘‘arcanum’’, he distinguished different grades of purity. The purest form was the most elaborated and also the most effective [27]. In the case of gold, the simplest one was ‘‘aurum potabile’’, also named ‘‘tinctura aurii’’ or ‘‘aurum vitae’’, characterized by an intense yellow color which could be administered orally. The second grade, with an intense red coloration, was the ‘‘oleum aurii’’, obtained by the concentration of ‘‘tinctura aurii’’ and was applied dropwise locally. The ruby-red ‘‘quinta essentia aurii’’ was the highest grade that gold could reach [26]. Some authors considered it as the direct precursor of the ‘‘lapis philosophorum’’ where the essence of gold was separated in high concentration by the metal in itself [22]. Starting with the first millennium a. C., ‘‘aurum potabile’’ had found quite a number of advocates in medicine, even if the recipes and indications varied. Around 1520, Paracelsus described the preparations of gold balsams starting from gold platelets [26, 28]. At the beginning of the eighteenth century, alchemical preparations were disputed: a considerable number of authors still believed that some of these medicines were highly effective, while others tried to debunk them with rationalistic arguments [29]. Some authors held that most aurum preparations had not really converted the rather inert metal into a salt [30]. In 1748,
Christoph Heinrich Keil reported the preparation of ‘‘tinctura solis’’ (like ‘‘aurum vitae’’) with an antimony arsenic sulfur powder as ‘‘tribus’’ (crafting material) [27]. The obtained product was administered orally after dissolution in spirit and had anodyne properties. In the seventeenth century, Bartholoma¨us Kretschmar made his recipe for ‘‘aurum vitae’’ public, which he advertised to be an efficient remedy against syphilis, dropsy, madness, epilepsy, malaria, icterus, leprosy, lupus, cancer (ulcers), skin rashes, fistulae, and cysts. He considered it also as a preservative (i.e., prophylactic medicine) against poisoning, magic and plague [31]. Preparation of a historical gold preparation and its biochemical evaluation Following our previous interest in the chemical biology of gold-containing drugs, we chose the mentioned historical receipt described by Kretschmar as a typical example of an early gold medicine to prepare ‘‘aurum vitae’’ and investigate its crucial biochemical effects [31]. For this purpose, the historic recipe, written in German of the seventeenth century, was translated into an English version and the medication was prepared in a standard equipped synthetic medicinal chemistry laboratory (see Fig. 1 and the supporting information for the historic text and its translation). In short, to prepare ‘‘aurum vitae’’, metallic gold and antimony sulfide were dissolved in ‘‘aqua regis’’ (royal
Fig. 1 Print of the cover page of ‘‘Vier chymischeTracta¨tlein’’ edited by Bartholoma¨us Kretschmar [31]
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Fig. 2 Isolated and dried ‘‘aurum vitae’’ product
water, HCl conc.:HNO3 conc./3:1), while mercury was dissolved in nitric acid 50 % (see the experimental section and the SI for details). The three solutions were mixed and heated under reflux conditions, then treated with ethanol and water and distilled repeatedly. The final ‘‘aurum vitae’’ was dried in a muffle oven and was obtained as a yellow– brown powder (see Fig. 2) in good agreement with the description reported in the ‘‘Vier Chymischen Tracta¨tlein’’ [31]. The used procedure represents a downscaled version of the historical recipe (e.g., starting from 0.15 g of gold instead of 15 g). Volumes were not indicated in the text by Kretschmar and were applied as appeared appropriate. The composition of aurum vitae was analyzed by atomic absorption spectroscopy and by elemental analysis (see supporting info). These measurements indicated a significant loss of mercury, most likely by evaporation during the preparation procedure. Relatively high amounts of chloride were detected, whereas sulfur was found in small amounts and nitrogen was absent. Overall, the analyses suggested that the metals are largely present in the form of a mixture of their chlorides and oxides. Until the nineteenth century, the understanding of cancer was not limited to malignant growth. Ulcers or benign tissue proliferation could also be denoted by this term. As mentioned above, ‘‘aurum vitae’’ was also prescribed as anticancer agent, yet there was no specific theory aiming to disclose the possible mechanism of action of this alchemic preparation. Although recent research has shown that a single mode of action is unlikely to exist, nowadays a preferential molecular target for gold-based drugs has been discovered: thioredoxin reductase (TrxR) [32, 33]. While a stringent direct correlation between gold biological activity and thioredoxin reductase activity in vivo is still missing, several recent works suggest that living cell systems treated with gold compounds displayed a strong onset of the antioxidant network, in which TrxR plays a central role [4, 5, 34–36]. TrxR is a key protein regulating cellular homeostasis and apoptosis. It is involved in various Table 1 TrxR and GR inhibition and antiproliferative effects in HT-29 and MCF-7 cancer cells triggered by the aurum potabile preparation and auranofin as reference
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pathological conditions and is overexpressed in cancer cells [34, 37]. Hence, we were interested in finding evidence that the historical preparation had a target and bioactivity comparable to modern gold drugs. In fact, the aurum vitae preparation strongly inhibited TrxR activity in the range of established drugs like auranofin (see Table 1) confirming an analogy between modern gold drugs and the historical alchemic preparation. Aurum vitae inhibited the activity of glutathione reductase (GR), which is an enzyme closely related to TrxR (but not a preferred drug target), at approximately threefold higher concentrations indicating only a moderate preference for TrxR inhibition. However, modern gold metallodrugs including auranofin can achieve a substantially higher selectivity for TrxR. [38–40]. It has to be noted here that the mercury and antimony ions contained in the preparation are also known to interfere with thiol- and selenol-containing enzymes and are likely to contribute largely to the overall biological activity. In particular, antimony-based agents are nowadays still used in the treatment of some leishmanial infections [41, 42]. The effects of ‘‘aurum vitae’’ on the proliferation of two cancer cell lines (HT-29 colon cancer and MCF-7 breast cancer) were also investigated (see Table 1), since the antiproliferative effects of gold and other metal-based agents are nowadays widely established facts [3, 4]. As expected, the preparation presented good antiproliferative effects in these two cancer cell lines, again in the range of auranofin.
Conclusions In conclusion, the results presented herein indicate that enzyme inhibition and cell growth inhibitory effects contribute to the pharmacological action of aurum vitae. It is thus very likely that the preparation triggered therapeutic effects in patients with tumors or infectious diseases. This might explain why gold-based medicines remained in pharmacopeas or even had a modest come back in nineteenth century, long before modern pharmacology formulated new explanations of the mode of action and possible therapeutic indications. However, from a pharmacological viewpoint, it must be assumed that patients treated with aurum vitae preparations suffered strong side effects related to antimony and mercury contained in this historical medicine.
Sample
TrxR IC50 (ng Au/mL)
GR IC50 (ng Au/mL)
HT-29 IC50 (lg Au/mL)
MCF-7 IC50 (lg Au/mL)
Aurum vitae
0.59 ± 0.24
2.07 ± 1.56
0.52 ± 0.05
0.94 ± 0.01
Auranofin
1.78 ± 0.00
2,953 ± 19.7
0.51 ± 0.08
0.22 ± 0.06
J Biol Inorg Chem Acknowledgments The literature research in history of pharmacy and the experimental contributions of Nuray Ates, Andre´ Morawetz, ¨ zen and Jan Carlos Quistorf are gratefully appreciated. Canan O
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24. Nowotny O (2004) Die Geschichte der Goldmedizin von der Antike u¨ber die Zeit der Alchemie bis zur Gegenwart. In: Meyer K (ed) Die Schelenz-Stiftung IV 1989–2003. Wiss. Verl.-Ges., Stuttgart, pp 99–112 25. Figala K (1998) Quintessenz, Alchemie. In: Claus P, Karin F (eds) Lexikon einer hermetischen Wissenschaft, pp 300–302 26. Paracelus T (1977) De morbis amentium, Bu¨cher und Schriften, ed. J. Huser, vol II, Hildesheim: Olms (reprint of the edition Basilea 1589) 27. Keil CH (1748) Compendio¨ses doch vollkommenes medicinischchymisches Handbu¨chlein, 6th edn. Martini, Leipzig 28. Paracelsus T (1971) De viribus membrorum interiorum. In: Huser J (ed) Bu¨cher und Schriften Edition, vol I. Olms, Hildesheim (reprint of the edition Basilea 1589), part 2: 5–6 29. Stahl GE (1728) Materia Medica. Zimmermann und Gerlach, Dresden 30. Kunckel J (1716) Collegium physico-chymicum experimentale, oder Laboratorium chymicum, Heyl, Hamburg, Leipzig 31. Kretschmar B (1977) Vier chymische Tracta¨tlein, VD17 39:139490P; Ferguson, Bibliotheca Chemica, vol 2, S. 509, pp 64–66. doi:10.3931/e-rara-5296 32. Bindoli A, Rigobello MP, Scutari G, Gabbiani C, Casini A, Messori L (2009) Thioredoxin reductase: a target for gold compounds acting as potential anticancer drugs. Coord Chem Rev 253:1692–1707 33. Gromer S, Arscott LD, Williams CH, Schirmer RH, Becker K (1998) Human placenta thioredoxin reductase, isolation of the selenoenzyme, steady state kinetics and inhibition by therapeutic gold compounds. J Biol Chem 273:20096–20101 34. Holmgren A, Lu J (2010) Thioredoxin and thioredoxin reductase: current research with special reference to human disease. Biochem Biophys Res Commun 396:120–124 35. Citta A, Schuh E, Mohr F, Folda A, Massimino ML, Bindoli A, Casini A, Rigobello MP (2013) Fluorescent silver(I) and gold(I)N-heterocyclic carbene complexes with cytotoxic properties: mechanistic insights. Metallomics 5:1006–1015 36. Sun RWY, Fong TTH, Li CKL, Yang ZF, Zou T, Siu AFM, Che CM (2013) A dinuclear cyclometalated gold(III)-phosphine complex targeting thioredoxin reductase inhibits hepatocellular carcinoma in vivo. Chem Sci 4:1979–1988 37. Arner ESJ, Holmgren A (2006) The thioredoxin system in cancer. Semin Cancer Biol 16:420–426 38. Gandin V, Fernandes AP, Rigobello MP, Dani B, Sorrentino F, Tisato F, Bjo¨rnstedt M, Bindoli A, Sturaro A, Rella R, Marzano C (2010) Cancer cell death induced by phosphine gold(I) compounds targeting thioredoxin reductase. Biochem Pharmacol 79:91–101 39. Hickey JL, Ruhayel RA, Barnard PJ, Baker MV, Berners-Price SJ, Filipovska A (2008) Mitochondria-targeted chemotherapeutics: the rational design of gold(I) N-heterocyclic carbene complexes that are selectively toxic to cancer cells and target protein selenols in preference to thiols. J Am Chem Soc 130:12570– 12571 40. Rubbiani R, Schuh E, Meyer A, Lemke J, Wimberg J, MetzlerNolte N, Meyer F, Mohr F, Ott I (2013) TrxR inhibition and antiproliferative activities of structurally diverse gold N-heterocyclic carbene complexes. Med Chem Commun 4:942–948 41. Haldar AK, Sen P, Roy S (2012) Use of antimony in the treatment of leishmaniasis: current status and future directions. Mol Biol Int. doi:10.4061/2011/571242 42. Baiocco P, Colotti G, Franceschini S, Ilari A (2009) Molecular basis of antimony treatment in leishmaniasis. J Med Chem 52:2603–2612
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ORIGINAL PAPER
Historical and biochemical aspects of a seventeenth century gold-based aurum vitae recipe Riccardo Rubbiani • Bettina Wahrig Ingo Ott
•
Received: 1 December 2013 / Accepted: 29 March 2014 Ó SBIC 2014
Abstract The medicinal chemistry and biomedical applications of gold complexes have been intensively studied over the last decades. Some complexes have been used for the treatment of rheumatoid arthritis, and a considerable number of new metallodrug candidates have been developed as new anticancer drugs and anti-infectives. However, the therapeutic use of gold and its complexes goes back to ancient times and was also of great importance for alchemists until the modern age. In this report, we give an overview of the alchemic medicine between the sixteenth and the early eighteenth century and describe the cytotoxicity and thioredoxin reductase (TrxR) inhibition of a typical ‘‘aurum vitae’’ medicine, which was prepared according to a recipe by Bartholoma¨us Kretschmar from the seventeenth century. ‘‘Aurum vitae’’ consists of a mixture of gold, mercury and antimony complexes and shows the expected cytotoxic and TrxR inhibitory properties providing some rationale for therapeutic effects of this kind of historical medicinal preparation.
Electronic supplementary material The online version of this article (doi:10.1007/s00775-014-1135-4) contains supplementary material, which is available to authorized users. R. Rubbiani I. Ott (&) Institute of Medicinal and Pharmaceutical Chemistry, Technische Universita¨t Braunschweig, Beethovenstraße 55, 38106 Braunschweig, Germany e-mail: [email protected] B. Wahrig Pharmazie- und Wissenschaftsgeschichte, Technische Universita¨t Braunschweig, Beethovenstraße 55, 38106 Braunschweig, Germany
Keywords Alchemy Antimony Aurum vitae Cytotoxicity Gold
Introduction The last decades have witnessed a steadily increasing interest in the biochemistry and biomedical applications of gold complexes [1]. Gold-based drugs such as Ridaura (auranofin), Myocrisin (aurothiomalate) and Solgonal (aurothioglucose) are nowadays used in the treatment of rheumatoid arthritis [2]. Moreover, gold(I) and gold(III) complexes have been demonstrating promising anticancer, antiviral and antiparasitic properties in many preclinical studies [3–7]. However, while most of the current understanding of the biomedical properties of gold species was established during the last century [8–10], gold preparations were already involved in the trades and in medicine more than 4,000 years ago [11, 12]. Gold-based remedies have been believed to be effective against a considerable number of diseases since ancient times and were also produced by many alchemists of medieval until modern ages [13–19]. Alchemy declined with the development of modern science, but many experimental and basic laboratory procedures are still recognizable today. However, historical preparations are not widely accessible today and the descriptions how to prepare those are related to pharmaceutical and biomedical concepts of the respective time. Accordingly, a reinvestigation of historical gold remedies appears not to have been performed so far. With this in mind, we report in this paper on historical aspects of medical gold preparations between the sixteenth and twentieth century and on selected biochemical key properties of a seventeenth century gold-based medicine, which we have prepared according to a historical procedure.
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J Biol Inorg Chem
Materials and methods General All solvents were used as received from Sigma-Aldrich or Fluka. Gold powder was purchased from ChemPur, mercury was purchased from Acros Organic, antimony(III) sulfide was purchased from Sigma-Aldrich.
concentration over time followed a linear trend (r2 C 0.99), and the enzymatic activities were calculated as the slopes (increase in absorbance per second) thereof. For each tested compound, the non-interference with the assay components was confirmed by a negative control experiment using an enzyme-free solution. The IC50 values were calculated as the concentration of compound decreasing the enzymatic activity of the untreated control by 50 % and are given as the means and errors of 3–6 independent experiments.
Aurum vitae preparation Antiproliferative experiments 0.150 g of gold (0.38 mmol) was dissolved in 5–10 mL of ‘‘aqua regis’’ (conc HNO3:conc HCl, 1:3) forming solution A. 0.750 g of mercury (1.87 mmol) was dissolved in HNO3 50 % giving solution B. 0.150 g of antimony sulfide Sb2S3 (0.44 mmol) was dissolved in ‘‘aqua regis’’ (conc HNO3:conc HCl, 1:3) forming solution C. Solution A, B and C were mixed and heated under reflux conditions and vigorous stirring for 12 h. The suspension was allowed to cool down and treated with 20 mL of ethanol absolute and 20 mL of distilled H2O. The obtained solution was distilled at 100 °C for 6 h and the distillate was discarded. A volume of 50 mL of ethanol 96 % was added, the solution was distilled at 100 °C and the distillate discarded. The last step was repeated 6 times. The finally obtained residue was placed in muffle furnace at 600 °C for 24 h and turned into a yellow–brown solid. Enzyme inhibition assay To determine the inhibition of TrxR and GR, an established microplate reader-based assay was performed [20]. For this purpose, commercially available rat liver TrxR or baker yeast GR (both from Sigma-Aldrich) were used and diluted with distilled water to achieve a concentration of 2.0 U/mL. The compounds were freshly dissolved as stock solutions in DMF. To each 25 lL aliquot of the enzyme solution, each 25 lL of potassium phosphate buffer pH 7.0 containing the compounds in graded concentrations or vehicle (DMF) without compounds (control probe) was added, and the resulting solutions (final concentration of DMF: max. 0.5 % v/v) were incubated with moderate shaking for 75 min at 37 °C in a 96-well plate. To each well 225 lL of reaction mixture (1,000 lL of reaction mixture consisted of 500 lL of potassium phosphate buffer, pH 7.0, 80 lL of 100 mM EDTA solution, pH 7.5, 20 lL of 0.05 % BSA solution, 100 lL of 20 mM NADPH solution, and 300 lL of distilled) water were added, and the reaction was started by the addition of 25 lL of an 20 mM ethanolic 5,50 -dithio-bis-nitrobenzoic acid (DTNB) solution. After proper mixing, the formation of 5-thio-nitrobenzoic acid (5-TNB) was monitored with a microplate reader (Perkin-Elmer VictorX4) at 405 nm in 10-s intervals for 6 min. The increase in 5-TNB
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The antiproliferative effects in MCF-7 and HT-29 cells after 72 h (HT-29) or 96 h (MCF-7) exposure to the gold preparations were evaluated according to a procedure already described in the literature [20]. For the experiments, the compounds were prepared freshly as stock solutions in DMF and diluted with the cell culture medium to the final assay concentrations (0.1 % v/v DMF). The cells were cultured in a 75 cm2 flask with 10 mL of cell culture medium. A volume of 100 lL of 12,000 cells/mL medium (MCF-7) or 6,000 cells/mL (HT-29) was seeded in 96-well plates and incubated for 48 (HT-29) or 72 h (MCF-7) at 37 °C under 5 % CO2. Afterward, the medium was removed and replaced with medium containing the substances in different concentrations. The cells were exposed to the complexes for 72 (HT-29) or 96 h (MCF-7). The medium was removed, and the cells were washed with PBS and stained with 100 lL of 0.02 % crystal violet for 30 min. The excess of crystal violet was removed, the plates were washed with water and dried, 180 lL of 70 % ethanol was added to each well, and the plates were read at 595 nm in a microplate reader (PerkinElmer VictorX4) after 3–4 h of gentle shaking. The IC50 values were calculated as the concentrations reducing proliferation of untreated control cells by 50 % and are given as the means and errors of at least two independent experiments (each performed with n = 6).
Results and discussion Overview of the alchemic medicine between the sixteenth and the early eighteenth century Alchemic preparations started with crude drugs, and they included often a large number of elaborate and time-consuming operations. These were considered to separate the beneficial properties from the toxic effects of a substance developing a kind of therapeutic essence. Gold played a key role within the alchemic tradition [21]. The deep interest of the alchemists in this element was related to their efforts to undertake material transformations of one element into the others with the final aim to transmute base
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metals into gold. In this context, the ‘‘lapis philosophorum’’ or philosopher’s stone was a mediating substance, which many alchemists sought to produce [22]. But although the philosopher’s stone was probably never created, the growing (al)chemical knowledge of specific procedures found many applications. Alchemy became a field of applied knowledge, may be even of ‘‘technoscience avant la lettre’’ [23]. Besides their close relationship to mining and smelting, glass and pottery production, alchemical substances found many medicinal applications [24]. Among them, gold-based remedies were often considered a sort of ‘‘panacea’’, a remedy for all or nearly all the diseases. Moreover, gold medicaments were thought to have rejuvenating properties because of the glittering yellow color and the comparison with the divinized ‘‘Sun’’ [11, 12, 21]. Before the alchemical era, gold was administered as solid gold leaves suspended in wine, ‘‘Spiritus’’ (distilled alcohol) and fruit or plants juice and this tradition was continued in official pharmacopeas and within pharmaceutical traditions outside alchemy [12, 25]. However, the alchemists came to the conclusion that a liquid gold preparation could have more chances to address a disease, because it would dissipate the noxious humors which stood at the origin of it. To achieve this status, the transformation of gold was essential. Philippus Theophrastus Aureolus Bombastus von Hohenheim (also called Paracelsus) elaborated both the concept of ‘‘arcanum’’ and gold preparations, among them ‘‘aurum potabile’’ which most likely existed already before him [26]. According to the theory of ‘‘arcanum’’, he distinguished different grades of purity. The purest form was the most elaborated and also the most effective [27]. In the case of gold, the simplest one was ‘‘aurum potabile’’, also named ‘‘tinctura aurii’’ or ‘‘aurum vitae’’, characterized by an intense yellow color which could be administered orally. The second grade, with an intense red coloration, was the ‘‘oleum aurii’’, obtained by the concentration of ‘‘tinctura aurii’’ and was applied dropwise locally. The ruby-red ‘‘quinta essentia aurii’’ was the highest grade that gold could reach [26]. Some authors considered it as the direct precursor of the ‘‘lapis philosophorum’’ where the essence of gold was separated in high concentration by the metal in itself [22]. Starting with the first millennium a. C., ‘‘aurum potabile’’ had found quite a number of advocates in medicine, even if the recipes and indications varied. Around 1520, Paracelsus described the preparations of gold balsams starting from gold platelets [26, 28]. At the beginning of the eighteenth century, alchemical preparations were disputed: a considerable number of authors still believed that some of these medicines were highly effective, while others tried to debunk them with rationalistic arguments [29]. Some authors held that most aurum preparations had not really converted the rather inert metal into a salt [30]. In 1748,
Christoph Heinrich Keil reported the preparation of ‘‘tinctura solis’’ (like ‘‘aurum vitae’’) with an antimony arsenic sulfur powder as ‘‘tribus’’ (crafting material) [27]. The obtained product was administered orally after dissolution in spirit and had anodyne properties. In the seventeenth century, Bartholoma¨us Kretschmar made his recipe for ‘‘aurum vitae’’ public, which he advertised to be an efficient remedy against syphilis, dropsy, madness, epilepsy, malaria, icterus, leprosy, lupus, cancer (ulcers), skin rashes, fistulae, and cysts. He considered it also as a preservative (i.e., prophylactic medicine) against poisoning, magic and plague [31]. Preparation of a historical gold preparation and its biochemical evaluation Following our previous interest in the chemical biology of gold-containing drugs, we chose the mentioned historical receipt described by Kretschmar as a typical example of an early gold medicine to prepare ‘‘aurum vitae’’ and investigate its crucial biochemical effects [31]. For this purpose, the historic recipe, written in German of the seventeenth century, was translated into an English version and the medication was prepared in a standard equipped synthetic medicinal chemistry laboratory (see Fig. 1 and the supporting information for the historic text and its translation). In short, to prepare ‘‘aurum vitae’’, metallic gold and antimony sulfide were dissolved in ‘‘aqua regis’’ (royal
Fig. 1 Print of the cover page of ‘‘Vier chymischeTracta¨tlein’’ edited by Bartholoma¨us Kretschmar [31]
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Fig. 2 Isolated and dried ‘‘aurum vitae’’ product
water, HCl conc.:HNO3 conc./3:1), while mercury was dissolved in nitric acid 50 % (see the experimental section and the SI for details). The three solutions were mixed and heated under reflux conditions, then treated with ethanol and water and distilled repeatedly. The final ‘‘aurum vitae’’ was dried in a muffle oven and was obtained as a yellow– brown powder (see Fig. 2) in good agreement with the description reported in the ‘‘Vier Chymischen Tracta¨tlein’’ [31]. The used procedure represents a downscaled version of the historical recipe (e.g., starting from 0.15 g of gold instead of 15 g). Volumes were not indicated in the text by Kretschmar and were applied as appeared appropriate. The composition of aurum vitae was analyzed by atomic absorption spectroscopy and by elemental analysis (see supporting info). These measurements indicated a significant loss of mercury, most likely by evaporation during the preparation procedure. Relatively high amounts of chloride were detected, whereas sulfur was found in small amounts and nitrogen was absent. Overall, the analyses suggested that the metals are largely present in the form of a mixture of their chlorides and oxides. Until the nineteenth century, the understanding of cancer was not limited to malignant growth. Ulcers or benign tissue proliferation could also be denoted by this term. As mentioned above, ‘‘aurum vitae’’ was also prescribed as anticancer agent, yet there was no specific theory aiming to disclose the possible mechanism of action of this alchemic preparation. Although recent research has shown that a single mode of action is unlikely to exist, nowadays a preferential molecular target for gold-based drugs has been discovered: thioredoxin reductase (TrxR) [32, 33]. While a stringent direct correlation between gold biological activity and thioredoxin reductase activity in vivo is still missing, several recent works suggest that living cell systems treated with gold compounds displayed a strong onset of the antioxidant network, in which TrxR plays a central role [4, 5, 34–36]. TrxR is a key protein regulating cellular homeostasis and apoptosis. It is involved in various Table 1 TrxR and GR inhibition and antiproliferative effects in HT-29 and MCF-7 cancer cells triggered by the aurum potabile preparation and auranofin as reference
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pathological conditions and is overexpressed in cancer cells [34, 37]. Hence, we were interested in finding evidence that the historical preparation had a target and bioactivity comparable to modern gold drugs. In fact, the aurum vitae preparation strongly inhibited TrxR activity in the range of established drugs like auranofin (see Table 1) confirming an analogy between modern gold drugs and the historical alchemic preparation. Aurum vitae inhibited the activity of glutathione reductase (GR), which is an enzyme closely related to TrxR (but not a preferred drug target), at approximately threefold higher concentrations indicating only a moderate preference for TrxR inhibition. However, modern gold metallodrugs including auranofin can achieve a substantially higher selectivity for TrxR. [38–40]. It has to be noted here that the mercury and antimony ions contained in the preparation are also known to interfere with thiol- and selenol-containing enzymes and are likely to contribute largely to the overall biological activity. In particular, antimony-based agents are nowadays still used in the treatment of some leishmanial infections [41, 42]. The effects of ‘‘aurum vitae’’ on the proliferation of two cancer cell lines (HT-29 colon cancer and MCF-7 breast cancer) were also investigated (see Table 1), since the antiproliferative effects of gold and other metal-based agents are nowadays widely established facts [3, 4]. As expected, the preparation presented good antiproliferative effects in these two cancer cell lines, again in the range of auranofin.
Conclusions In conclusion, the results presented herein indicate that enzyme inhibition and cell growth inhibitory effects contribute to the pharmacological action of aurum vitae. It is thus very likely that the preparation triggered therapeutic effects in patients with tumors or infectious diseases. This might explain why gold-based medicines remained in pharmacopeas or even had a modest come back in nineteenth century, long before modern pharmacology formulated new explanations of the mode of action and possible therapeutic indications. However, from a pharmacological viewpoint, it must be assumed that patients treated with aurum vitae preparations suffered strong side effects related to antimony and mercury contained in this historical medicine.
Sample
TrxR IC50 (ng Au/mL)
GR IC50 (ng Au/mL)
HT-29 IC50 (lg Au/mL)
MCF-7 IC50 (lg Au/mL)
Aurum vitae
0.59 ± 0.24
2.07 ± 1.56
0.52 ± 0.05
0.94 ± 0.01
Auranofin
1.78 ± 0.00
2,953 ± 19.7
0.51 ± 0.08
0.22 ± 0.06
J Biol Inorg Chem Acknowledgments The literature research in history of pharmacy and the experimental contributions of Nuray Ates, Andre´ Morawetz, ¨ zen and Jan Carlos Quistorf are gratefully appreciated. Canan O
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