Cangrande della Scala :: Risultati della ricerca

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Cangrande della Scala

INDICE DEI CONTENUTI:

- La mummia di Cangrande della Scala, signore di Verona

- Risultati della ricerca

ALTRE PAGINE DELLA SEZIONE
"Laboratorio"

VAI ALLA PAGINA INTRODUTTIVA

Gino fornaciari a, b, Valentina Giuffra a, b, *, federica Bortolotti c, Rossella Gottardo c, Silvia Marvelli d, Marco Marchesini e, Silvia Marinozzi f, Antonio fornaciari a, b, Giorgio Brocco g, franco Tagliaro c

a Division of Paleopathology, Department of Translational Research on New Technologies in Medicine and Surgery, University of Pisa, Via Roma 57, 56126 Pisa, Italy
b Center for Anthropological, Paleopathological and Historical Studies of the Sardinian and Mediterranean Populations, Department of Biomedical Sciences, University of Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy
c Unit of forensic Medicine, Department of Public Health and Community Medicine, University of Verona, P.le L.A. Scuro 10, 37134 Verona, Italy
d Palynological Laboratory, Archaeoenvironmental Laboratory, C.A.A. Giorgio Nicoli, Via Marzocchi 17, 40017 San Giovanni in Persiceto, Italy
e Superintendence to Archaeological Properties of Emilia-Romagna, Via Belle Arti, 52, 40126 Bologna, Italy
f Section of History of Medicine, Department of Experimental Medicine, University of Rome “La Sapienza”, Viale dell'Universita' 34a, 00185 Rome, Italy
g Section of Biological Chemistry, Department of Life and Reproduction Sciences, University of Verona, Strada Le grazie 8, 37134 Verona, Italy

 

KEY-WORDS: Middle Ages Natural mummy foxglove Digitalis Palynology Toxicology Digoxin Digitoxin

 Abstract
The natural mummy of Cangrande della Scala was exhumed from its tomb in the church of Santa Maria Antiqua in Verona and was submitted to a multidisciplinary study, that included archaeological, palaeopathological, palynological, toxicological and historical investigation. The body of Cangrande, still wearing his precious clothes, was in a good state of preservation. Palynological analyses demonstrated the presence of pollen grains of Digitalis sp./foxglove in the rectum content, along with Matricaria chamomilla/chamomille and Morus nigra/black mulberry. Toxicological analyses showed toxic concentrations of digoxin and digitoxin, two Digitalis glycosides, in the liver and faeces samples. Both palynological and toxicological data suggest an intoxication through the oral administration of an infusion or decoction of leaves and flowers of Digitalis.
Cangrande died on July 22 1329, four day after his triumphal entrance in the city of Treviso. The sudden death was preceded by vomit and diarrhoea with fever that, according to written documents, he had contracted a few days before by “drinking from a polluted spring”. The gastrointestinal symptoms manifested by Cangrande in his last hours of life are compatible with the early phase of Digitalis intoxication and the hypothesis of poisoning is mentioned by some local historical sources. The palaeopathological analyses confirm a Digitalis poisoning. The most likely hypothesis on the causes of death is that of a deliberate administration of a lethal amount of Digitalis. Although several cases of poisoning through the use of organic substances are known from historical sources, no other direct evidences are documented in the palaeopathological literature.


  • Introduction

Cangrande della Scala (1291e1329) is the most celebrated member of the Scaligeri dynasty, which ruled Verona from 1277 to 1387 (Varanini, 1988; Spangenberg, 1993).
Leading patron of the poet Dante Alighieri, who composed a letter (Epistola XIII) in honour of his host in Verona (Branca, 1907), Cangrande was a great warrior and an important autocrat of his time. After the death of his brother Alboino he became the sole ruler of Verona in 1311, at the age of twenty. Cangrande was regarded as the leader of the Ghibelline party in Northern Italy, bringing the city of Vicenza under his control (1314). After several years of intermittent and brutal conflicts he took possession of Padua (1328) and Treviso (1329).
On 18 July 1329 the entrance of Cangrande into the city of Treviso represented the crowning achievement of his long struggle to submit the entire region of Veneto (Northern Italy). However, his triumph was marred by the fact that he had become seriously ill as a result of vomit and diarrhoea, which he had contracted a few days before by “drinking from a polluted spring”, according to contemporary accounts (Varanini, 2004). Cangrande died on the morning of July 22.
The body of Cangrande was temporarily housed in the church of Santa Maria Antiqua in Verona, but it then appears to have been moved twice, first to a marble tomb in the churchyard and secondly to the monumental marble tomb over the church entrance, above which there is an equestrian statue of Cangrande in tournament armour (fornaciari, 2004; Napione, 2006) (fig. 1).
In february 2004 the tomb of Cangrande was opened to allow a multidisciplinary study of the corpse, which included archaeological, palaeopathological, palynological, toxicological and historical investigation. The natural mummy, still wearing its precious clothes, appeared in good state of preservation (Napione, 2006).
The aim of this paper was to investigate the causes of Cangrande's death, taking also into consideration that rumours of poisoning are reported by traditional chronicles (Varanini, 2004).

  • Materials and methods
    • Palaeopathological study

The mummy of Cangrande (fig. 2) was submitted to autopsy (fig. 3a), performed through a circular opening of the abdomen, from  the  sternum  to  the  pubis,  which  allowed  to  reach  the abdominal and thoracic cavities (fornaciari, 2004, 2006). The viscera appeared to be collapsed on the posterior wall and were identified according to their topographical position. Macroscopic, radiological and histological studies were performed. The latter included rehydration with Sandison solution (Sandison, 1955), routine paraffin-embedding and standard haematoxylin-eosin and Van Gieson's staining procedures.

    • Palynological analysis

Samples for palynological analyses were taken from two internal regions of Cangrande's mummy, the left hemi-abdomen (colon) and the rectum content (faeces). The analyses were carried out applying an already tested analytical approach (Giuffra et al., 2011). The method includes the following phases: addition of a tablet of Lycopodium spores re-suspended in HC1 10% for the calculation of pollen concentration (¼number of pollen grains/gram); treatment with acetic acid to dehydrate the preparation; Erdtman acetolysis; addition of 40% of Hf without pre-heating for 24 h; washings in distilled water; addition of ethanol; desiccation in thermostatic oven; preparation of glycerine jelly-fixed slides. The observation of the samples was performed at 1000x light microscope magnification (ocular 10x and objective 100x) with the help of keys, atlases and a reference pollen collection. In particular, the identification of the Digitalis pollen was obtained by using a manual (Moore et al., 1991), whereas that of Digitalis cf. purpurea was based on the comparison with the laboratory collection of the authors.
The pollen terminology is based on Berglund and Ralska-Jasiewiczowa (1986). The botanic terminology follows Pignatti (1982).

    • Toxicological analysis

Toxicological analyses were performed on different samples taken from the mummy of Cangrande to detect any traces of poisons or toxic substances or to find evidences of any possible pharmacological treatments, which could explain his rapid death. In particular, the toxicological analyses were carried out on a sample of hair (100 mg) from the head vertex, two samples of rectum content (approximately 5 g each), and two liver samples (approximately 5 g each), which have been collected during the autopsy.
The extracts of the hair sample, of one faeces sample, and of one liver sample underwent screening toxicological analyses by using CE and HPLC-MS methods.
The CE analyses were performed on a P/ACE-MDQ capillary electropherograph equipped with a diode-array detector operating at 214 nm (Beckman Coulter, fullerton, CA, USA). Separations were carried out at 25 oC in an uncoated fused-silica capillary (Composite Metal Service Worcester, UK), with an internal diameter of 50 mm and a total length of 60 cm, using a buffer composed of 100 mM phosphate (pH 2.38) (Hudson et al., 1995).
The HPLC-MS analyses were performed on a 1100 HPLC coupled with a MSD-ion trap mass spectrometer, model SL (Agilent Technologies, Palo Alto, CA) using a Zorbax Eclipse XDB (2.1 x 150 mm, 5 mm particle size, Agilent Technologies) with gradient elution from 5% to 95% of solvent B lasting 20 min (solvent A: formic acid 0.1%; solvent B: methanol). The spray voltage was set at 4 kV and the skimmer at 40 V. for peak identification, the ion trap mass spectrometer was set to perform MS2  on the molecular ion of the selected compounds, using helium as collision gas.
The results of screening analyses were confirmed by using a 1100 HPLC coupled with a quadrupole Time of flight Mass Spectrometry (Q-TOf MS) Ultima (Waters MS Technologies, Manchester, UK). The separation was performed using a Synergi Hydro column 150 x 4.6 mm (Phenomenex, Torrance, CA) with gradient elution from 5% to 95% of solvent B lasting 50 min (solvent A: ammonium formate 10 mmol, pH 5; solvent B: acetonitrile). Electrospray ionization, in positive mode, was used. The HPLC flow was split (1:5) between the mass spectrometer and the waste. Analytes were identified on the basis of charged molecular ions, mass accuracy, and their collision-induced dissociation fragmentation.
A liver sample and a faeces sample underwent immunochemical analyses in order to determine digoxin and digitoxin. The samples were pulverised and rehydrated with distilled water in a proportion of 50:50. After 24 h, the samples were submitted to ultrasonication (35 kHz) for 3 min and then centrifuged for 15 min a 1500 x g. The determination of digoxin was carried out by using different antisera [Digoxin   IMMULITE®    (Diagnostic   Products   Corporation,   Los Angeles, USA); Digoxin Tina-quant® (Roche Diagnostic GmbH, Mannheim, Germania); Digoxin Assay Emit® 2000 (Syva Company, Dade Behring Inc., Cupertino, USA)] to minimize the risk of errors caused by cross-reactions. The determination of digitoxin was carried by using IMMULITE/IMMULITE® 1000 (Diagnostic Products Corporation, Los Angeles, USA).

Figura 1 Figura 2 Figura 3

 

  • Results
    • Palaeopathological  study

The palaeopathological study revealed that Cangrande suffered from mild osteoarthritis of the column, elbows and hips, with a meniscus calcification of the knees, probably related to the strong intense physical activity of the Prince. The liver was well recognizable, showing a typical anatomic shape, with a transversal diameter of 30 cm, both at macroscopic examination (fig. 3b) and at Computed Tomography (CT) (fig. 4). The presence of faeces in the rectal ampulla was observed (fig. 4). Histology demonstrated small atheromatous plaques on the luminal surface of the aorta, as well as a  picture  of  severe  anthracosis,  irregular  emphysema  and  fibrohyaline  nodular  formations,  probably  attesting  the  results  of tuberculosis, at the level of lungs.

    • Palynological analysis

Palynological analysis includes 4 taxa: 3 of Spermatophyta (1 trees/shrubs and 2 herbs) and 1 taxon of Pteridophyta. In the left hemi-abdomen (524 pollen grains/g) 2 taxa, Matricaria chamomilla (chamomile) (83.3%) and Morus nigra (black mulberry) (16.7%) were found. In the rectum (863 pollen grains/g) large amounts of Matricaria chamomilla (88.9%) (fig. 5a) and few pollen grains of Digitalis (foxglove) were identified (11.1%) (fig. 5b).
It was difficult to determine whether the pollens belonged to Digitalis purpurea L. or Digitalis lanata Ehrh. Indeed, the scarce amount of plant material found in the mummy prevented the use of DNA barcoding, a genetic technique already used for ancient plant remains to individuate without doubts the taxonomic identity of pollen elements (Gismondi et al., 2012, 2013). However, the morphological characteristics suggested that the pollens could belong to Digitalis cf. purpurea. This plant was largely cultivated in the past centuries with medicinal purposes. Nowadays it is widespread as ornamental plant, while several wild populations are known in woody glades and mountain pastures of the Alps and the spontaneous plant is present in Corse and Sardinia (Pignatti, 1982).

    • Toxicological analysis

The capillary electrophoretic and HPLC-MS analyses evidenced in the hair traces of harman and santonin, which are compounds contained in Passiflora sp. L. (passionflower) and Artemisia sp. L. (wormwood), respectively. Santonin was also found in the faeces and in the liver samples. Both Passiflora and Artemisia are historically well-known medical plants, which were used for their pharmacological properties. The presence of these substances in the hair could be explained by the administration of an external treatment or by the oral administration of a concoction. The latter possibility would be supported by the identification of santonin in the faeces and in the liver. Nevertheless, no one of these com pounds shows a considerable toxicity.
Immunochemical analyses of faeces and liver samples showed significant quantities of compounds immunoreactively similar to digoxin and digitoxin, which are two glycosides of Digitalis. The two glycosides were identified in toxic concentrations, in order of tens of ng/g, as showed in Table 1.

 

Digoxin-DPC

Digoxin Roche

Digoxin Syva

Digitoxin-DPC

Liver (ng/g)

14.0

5.6

16.8

62.4

Rectal feces (ng/g)

15.6

7.2

11.2

41.6

Table 1 - Concentration values of digoxin and digitoxin in the liver and feces of Cangrande.

 

  • Discussion

Several palynological studies, which were carried out over the past decades on the graves of important historical personages, have helped obtain valuable information on the intake of vegetable food and beverages consumed before the death, ingestion of toxic or poisonous substances, use of particular aromatic substances in funerary rites, origin of the clothes found in the burial, and vegetation of a particular range (Paganelli, 1981; Bertolani Marchetti and Mariotti Lippi, 1993; Scannerini, 1997; Paganelli, 2003; Marchesini and Marvelli, 2006; Piombino Mascali et al., 2013).
In the case of Cangrande, palynological spectra, although at low concentration values (102e103 pollen grains/gram), showed the intake of vegetal components by the Prince in the last moments of his life. In particular, they showed the ingestion of foxglove leaves and flowers.
Some  papers  are  published  on  the  significance  of  low  taxa pollen spectra and low values of entomophilous plants in archaeobotanical (Mercuri, 2008) and paleoforensic cases (Piombino Mascali et al., 2013). In the case of Cangrande the prevalent ingestion of foxglove leaves instead of flowers could explain the low concentration values of pollen grains.
Toxicological analyses have shown the presence of two active principles of Digitalis, digoxin and digitoxin, in liver and faeces samples. The concentrations of Digitalis glycosides determined in these experiments after the rehydration of samples in a ratio of 50/ 50 are in the toxic range (Baselt, 2008). However, taking into account a reasonably high loss of the compounds during the seven centuries elapsed since Cangrande's death, it is possible to infer that the digoxin and digitoxin concentrations measured in the tissues of the Prince at death time were well above the lethal concentrations.
An integrated evaluation of the experimental data provided by palynological and toxicological study clearly suggests an intoxication through the oral administration of an infusion or decoction of leaves and flowers of Digitalis. Autopsy could not reveal any alteration referable to Digitalis intoxication because acute poisoning does not leave traces at macroscopic level.
Digitalis poisoning is characterized by gastrointestinal symptoms including nausea, vomiting, diarrhoea, abdominal pain; central nervous manifestations, in particular somnolence, hallucinations, delirium and severe headache; effects on the heart like abnormal rhythms, the most serious of which is ventricular tachycardia, that can cause heart block (Bayer, 1991).
The gastrointestinal symptoms manifested by Cangrande in his last hours of life and described by historical sources are compatible with the early phase of Digitalis intoxication. The symptomatology of Cangrande includes vomiting and diarrhoea with acute pain (“fluxus ventris”, “fluxu obiit”, “corporeus fluxus stomachique dolor acutus”); sometimes there is reference to fever (“fluxum et febrem continuam ob laborem exercitus”) (Varanini, 2004).

Figura 4 Figura 5

It is difficult to ascertain whether the Digitalis fatal intoxication
of Cangrande was an intentional murder or a terrible mistake. The therapeutic properties of foxgloves were discovered in the second half of 18th century (Doyle, 2009), but the toxic effects of the plant must have been known much earlier. Unfortunately there is a gap in the documentary sources, since references to foxglove as a poison were not found before Renaissance times. Even if foxglove grew over the most part of Europe, it was never mentioned by Dioscorides, Theophrastus or other ancient writers.
One of the first historical source on the Digitalis is a manuscript copy of the Herbarium Apuleii Platonici, written at Bury St. Edmunds around 1120, where the English word foxglove appears along with the names of other plants prescribed to compose topical ointments for the treatment of wounds (Groves and Bisset, 1991). According to the medical and botanical literature from Roman to Modern age, all these plants are described and defined as poisonous, and employed as analgesics, hypnotics and narcotics only in small doses (Mattioli, 1563; Paulus Aegineta, 1846; De Renzi, 1853; Galenus, 1964e5; Plinius Secundus, 1969e1974; Theophrastus, 1980; McVaugh, 2008).
The sudden disappearance of a powerful and relatively young man probably favoured the  circulation of rumours about this death. Although some diseases are occasionally mentioned, such as the imprecise illnesses of 1315 and 1325, Cangrande was healthy, despite the  great physical efforts  during the  military campaigns of the last twenty years of his life. The hypothesis of poisoning is mentioned by some local historical sources, even if the documents are not anterior to the second half of the 14th century. A vernacular Venetian chronicle written soon after 1354 and the chronicler Galeazzo Gatari in the last part of the 14th century report that Cangrande was poisoned (Varanini, 2004). The Veronese notary Boninsegna da Mizzole, author of a local chronicle between 14th and 15th century, and the notary and historian Torello Saraina in the 15th century claimed that the poison had been hidden in a fruit (Varanini, 2004). On the other hand, poisoning was a diffused practice in the European Middle Ages (Collard, 2003).
Some details about poisoning modalities are reported  in  the 15th century by a Treviso chronicler (Anonimo foscariniano), who claims that the physician accused of poisoning had been imprisoned and hung after the confession. This dubitative and late version seems to be confirmed by a more authoritative source, the jurist and chronicler Guglielmo Cortusi, who was ambassador in Verona in 1328 and close to the environment of Alberto II della Scala (1306e1352), co-ruler of Verona after the death of Cangrande together  with  his  brother  Mastino  II  (1308e1351);  according  to Cortusi, who is silent about the causes of Cangrande's death, his physician died on the gallows (Varanini, 2004).
Considering that the therapeutic properties of Digitalis were recognized only in Modern times (fuchs, 1549),  in the case  of Cangrande an accidental ingestion of a vegetal mixture including flowers and leaves of Digitalis should not be excluded; glycoside ingestion probably occurred as a result of an erroneous identification of leaves, not always easy to distinguish from those of other edible plants, as occurs also today. Accidental poisonings consequent to ingestion of herbal concoctions containing foxglove leaves are reported in modern clinical literature (Dickstein and Kunkel, 1980; Bain, 1985; Maffe' et al., 2009; Lin et al., 2010; Mitchell, 2010; Castello et al., 2012).
But the most likely hypothesis is that of a deliberate administration of a lethal amount of Digitalis; the poison may have been masked in a decoction containing chamomile and black mulberry, prepared for some indisposition of Cangrande. Although several cases of poisoning through the use of organic substances are known from historical sources (Smith, 1952), no other direct evidences are documented in palaeopathological literature.

  • Conclusions

The multidisciplinary study performed on the natural mummy of Cangrande della Scala supports the hypothesis that Cangrande was victim of digitalis poisoning. Palynological analyses demonstrated the presence of foxglove as pollen grains in the rectum content and toxicological analyses confirmed the presence of two active principles of Digitalis, digoxin and digitoxin, present in toxic concentration in the liver and faeces. Although it is not possible to rule out totally an accidental intoxication, the most likely hypothesis is that of a deliberate administration of a lethal amount of Digitalis. It is more problematic to detect the instigator of the murder. The principal suspects are the neighbouring states, the Republic of Venice or Ducate of Milan, worried about the new regional power of Cangrande and Verona; at the death of Cangrande also his ambitious nephew Mastino, who became ruler of Verona in association with his brother Alberto, cannot be totally excluded as instigator.
The case of Cangrande represents the unique direct evidence of poisoning through the use of organic substances in the palaeopathological  literature.

  • Acknowledgements

We would to thank Prof. Daniela fausti (University of Siena), Alain Touwaide (Smithsonian Institution e National Museum of Natural History of Washington) and Andrea Ubrizsy Savoia (University of Rome “La Sapienza”) for providing important information about the historical research to identify foxglove in ancient medical and naturalist literature. Moreover, we would thank Dr. Silvia Martinucci (GSK Verona) for the confirmation analysis of santonin and harman, and Prof. Giacomo Gortenuti (University of Verona) for the imaging study.
A special thank to Dr. Paola Marini, Director of the Castelvecchio Museum, Verona.
This work was supported by a grant from the ARPA foundation (www.fondazionearpa.it).


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Svelate le cause della morte di Cangrande della Scala

L'equipe di (leggi tutto...)
Inserito il: Il 24/01/2015

Application of segmentation and 3D rendering techniques in the mummies of Cangrande della Scala (1291-1329) and S. Giacomo della Marca (1391-1476)

Computed Tomography (CT) has become a routinely non-invasive technique in paleopathology,... (leggi tutto...)
Inserito il: Il 10/01/2015

BACHECA DEI COMMENTI
  • Scavo archeologiaco di Badia PozzeveriEsplora i risultati dello scavo e le straordinarie scoperte
  • Scavo archeologico di BenabbioUn castello medievale e il suo cimitero
  • CangrandeCome morì il signore di Verona?
  • Giovanni dalle Bande Nere e Maria SalviatiIl più grande condottiero del XVI secolo. Cosa cè dietro la sua morte?
  • Ilaria del Carretto e la Cappella GuinigiCosa si cela dentro il monumento funebre di Jacopo Della Quercia?
Scavo archeologiaco di Badia Pozzeveri1 Scavo archeologico di Benabbio2 Cangrande3 Giovanni dalle Bande Nere e Maria Salviati4 Ilaria del Carretto e la Cappella Guinigi5
Summer schoolsProgetto Mummie SicilianeSegui gli scavi di BenabbioFrancesco IMaster in Bioarcheologia, paleopatologia e Antropologia Forense