Biotechnic & Histochemistry

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Brazilwood, sappanwood, brazilin and the red dye

brazilein: from textile dyeing and folk medicine to
biological staining and musical instruments

Rw Dapson & Cl Bain

To cite this article: Rw Dapson & Cl Bain (2015) Brazilwood, sappanwood, brazilin
and the red dye brazilein: from textile dyeing and folk medicine to biological
staining and musical instruments, Biotechnic & Histochemistry, 90:6, 401-423, DOI:
10.3109/10520295.2015.1021381

To link to this article: http://dx.doi.org/10.3109/10520295.2015.1021381

Published online: 20 Apr 2015.

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 Brazilwood, sappanwood, brazilin and the red dye
brazilein: from textile dyeing and folk medicine to
biological staining and musical instruments
RW Dapson1, CL Bain2
1Dapson & Dapson, LLC, Richland, Michigan and 2Mineralized Tissue Histology Research Laboratory, School of
Dentistry, Indiana University-Purdue University-Indianapolis, Indianapolis, Indiana

Accepted February 17, 2015

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Abstract

Brazilin is a nearly colorless dye precursor obtained from the heartwood of several species of trees
including brazilwood from Brazil, sappanwood from Asia and the Pacific islands, and to a minor
extent from two other species in Central America, northern South America and the Caribbean
islands. Its use as a dyeing agent and medicinal in Asia was recorded in the 2nd century BC, but
was little known in Europe until the 12th century AD. Asian supplies were replaced in the 16th
century AD after the Portuguese discovered vast quantities of trees in what is now Brazil.
Overexploitation decimated the brazilwood population to the extent that it never fully recovered.
Extensive environmental efforts currently are underway to re-create a viable, sustainable
population. Brazilin is structurally similar to the better known hematoxylin, thus is readily oxidized
to a colored dye, brazilein, which behaves like hematein. Attachment of the dye to fabric is by
hydrogen bonding or in conjunction with certain metallic mordants by coordinative bonding. For
histology, most staining procedures involve aluminum (brazalum) for staining nuclei. In addition
to textile dyeing and histological staining, brazilin and brazilein have been and still are used
extensively in Asian folk medicine to treat a wide variety of disorders. Recent pharmacological
studies for the most part have established a scientific basis for these uses and in many cases have
elucidated the biochemical pathways involved. The principal use of brazilwood today is for the
manufacture of bows for violins and other stringed musical instruments. The dye and other
physical properties of the wood combine to produce bows of unsurpassed tonal quality.

Key words: brazilein, brazilin, brazilwood, ethnopharmacology, musical instrument woods,

natural dyes, pernambuco, sappanwood, tropical forest conservation, violin bows

Brazilin is a red dye precursor obtained from the
heartwood of several species of tropical hardwoods,
principally brazilwood and sappanwood. While
several other natural dyes (cochineal/carmine,
indigo, kermes/kermesic acid and madder/alizarin)
have played pivotal roles as colorants through
history and in society (Balfour-Paul 2006, Greenfield
2005), brazilins influence and that of its source
Correspondence: Richard W. Dapson, Dapson & Dapson, LLC,
6951 East AB Avenue, Richland, MI 49083. Phone: 269-929-
7741, e-mail: dick@dapsons.com
2015 The Biological Stain Commission
Biotechnic & Histochemistry 2015, 90(6): 401423.
woods has been much broader, but lesser known.
We characterize here brazilin and its various sources.
We provide also an historical and geographical
review, and discuss environmental issues, chem-
istry, medicinal uses, pharmacology, and its role
in music, textile dyeing and histological staining.
Several important references in Portuguese are
reviewed here, which makes their information avail-
able to English speaking readers for the first time.
To clarify, brazilin, like hematoxylin, technically
is not a dye; it is colorless and has no real means of
binding to substrates. Nevertheless, both substances
have been referred to as dyes even in fields like
histology where the distinction is fairly well known.

DOI: 10.3109/10520295.2015.1021381 401

 Histologists buy or make hematoxylin solutions,
then stain nuclei with hematoxylin, when in fact
the active ingredient is hematein. In similar fash-
ion, early users of brazilin for histology referred to
its staining solutions as brazilin. Modern craft
dyers sometimes call their solutions brazilin rather
than brazilwood even when the dye bath is red
(obviously both brazilin and brazilein are pres-
ent). Here we prefer to refer to brazilin as a red dye
precursor.
Our article primarily is a review of literature;
however, some new information is presented in the
section on dye chemistry that is based on the use
of the molecular modeling software package, Chem
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Draw, as described elsewhere by Dapson (2005,
2013). We have drawn a structural formula in which
the energy of bonds between atoms is minimized
to achieve the lowest energy conformation, then
we performed quantum mechanical calculations to
obtain various numerical parameters that are use-
ful for characterizing the molecule and its chemical
behavior.
Nomenclature
Mohr (1950) posited that brasil is a corruption
of some unknown and lost word in an unidenti-
fied Asian language. Mayer (1943) derived the
word from the Arabic term, braza, which means
fiery red. In Europe, Portuguese traders and mer-
chants referred to the dye extracted from Asian
sappanwood as braise, meaning live or burning
ember. Various medieval names for the dye of com-
merce (brezellum, brasilium, bresellum as well as
the most common brasil) were derived from one
or several of these sources. Venetian tradesmen
referred to the dye as verzino (Italian) before 1500,
but when Portugal imported vast quantities from
South America, its name (brasil and its derivatives)
quickly superseded the Italian term. Whatever the
original derivation, the Portuguese named the land
from which they obtained the wood, Terra de Brasil,
later shortened to Brasil (Brazil in English), in rec-
ognition of this valuable resource.
Pernambuco (in both Portuguese and English)
is a geographic variety of brazilwood. The name is
derived from the state in Brazil from which some of
the finest wood was harvested for the manufacture
of bows for stringed instruments.
Brazilin (CI 75280, natural red 24; brasilina in
modern Portuguese) is the name used for histol-
ogy and pharmacology. In other fields, however, it
is more commonly called by the same name as the
trees from which it is derived, thus brazilwood or
brasilwood (pau-brasil in Portuguese, bois de Brsil in
402 Biotechnic & Histochemistry 2015, 90(6): 401423
French, both meaning wood of Brazil) for the main
South American source, and sappanwood or Sappan
Lignum from Southeast Asia and Malaysia. Other
names are given in Table 1. In most cases today,
brazilin is the technical term for reasonably pure
dye and the other names commonly are applied to
crude extracts and unextracted sawdust or wood
chips.
Historical and modern literature is quite con-
fusing because scientific, common and commercial
names of the source trees have been used rather
indiscriminately or inaccurately. Moreover, there
is little uniformity in spelling in English language
references, especially those from the middle 1800s
and earlier. Carelessness, transliteration from other
languages or the direct use of foreign terms in
English writings have made it difficult for modern
readers to know the true source of the dye.
On one occasion, confusion about nomencla-
ture led to a lawsuit, Seixas and Seixas vs. Woods
(New York State Supreme Court, 1804). Brazilleto
(brazilwood) was advertised and sold by a trad-
ing company, but the buyer later discovered that
the goods actually were peachum (peachwood,
Nicaragua wood). The former was a valuable
commodity, but the latter essentially was worth-
less. The trader refused to refund the money and
the buyer sued. The Court determined (with
dissension) that the trader was ignorant of the true
nature of the wood, did not knowingly deceive the
buyer, offered no warranty and thus was not held
liable. Incidentally, this became case law for caveat
emptor (let the buyer beware).
Brazilein is the scientific term for oxidized
brazilin. To avoid confusion with brazilin when
speaking, brazilein should be pronounced with
four syllables, while the Brazilian Portuguese word
brasileina is pronounced with five syllables. The
vernacular equivalent in textile usage was aged
brazilwood, aged sappanwood or aged Nicaragua
wood.
Sources of the dye
Brazilin is found in the heartwood of several species
of trees found in a tropical band across Central
and South America, India, and the Asian Pacific
coasts and islands (Table 1). The species of his-
torical and commercial importance are Caesalpinia
echinata, C. sappan and Haematoxylum brasiletto.
These and several closely related species are not
readily distinguished from one another with cer-
tainty by persons other than experienced botanists,
which further confounds the literature. All belong
to the legume family, Fabaceae (Leguminosae or
 Table 1. Common and scientific names of trees producing brazilin.
Scientific name Location
Caesalpinia echinata Brazil
Caesalpinia sappan Southeast Asia
Caesalpinia violacea Caribbean islands
(  C. brasiliensis)
Hematoxylum brasiletto Central America, northern Peachwood, peachum or peachum
South America
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Caesalpiniaceae according to some botanists).
Leaves of these three species are compound, con-
sisting of three to more than 10 branches (pinnae),
each of which in turn bear from two to 30 leaflets
(Fig. 1). The number of pinnae and the number of
leaflets on each pinna is highly variable within each
species depending upon geographic location and
probably genetic factors. Prickles are found at the
leaf bases. The trunk and branches may bear thorns.
Sweetly scented yellow flowers are clustered into
pannicles borne on the tips of branches or less often
at the bases of branches. Authorities differ concern-
ing the features that distinguish the species (cf.
Cardon 2007 and Cavalcante de Lima and Werneck
da Cunha 2013) and taxonomic revisions continue.
For our purposes, scientific and vernacular names
are as specified in Table 1.
Before overexploitation by Europeans, brazil-
wood trees (C. echinata) could reach up to 30 m
in height and 0.7 m in diameter (Cardon 2007).
Because of massive overexploitation in the 15th and
Fig. 1. Compound leaf of brazilwood measuring
approximately 32 cm in length. Photograph taken in 2014
in a park in Sao Paulo, Brazil.
Common name(s)
Brazilwood, pau-brasil (Port.),
pernambuco, brazilletto
Sappanwood, Lignum Sappan,
Sappan Lignum, lima wood, bois
de sappan (Fr.), suwo (Jap.)
Brazilette and brsillet (Sp.)
wood, Nicaragua wood, palo de
brasil (Sp.)
16th centuries, the species is most often a shrubby
tree that grows up to 12 m in height. The trees favor
dry, calcareous clay or sandy-clay soils from high,
dry areas to seasonally dry forests along the coast,
which is quite different from their close relative,
logwood (Haematoxylum campechianum), which is
found in lowland swamps along the coast of Belize
and northern Yucatan (Mexico).
Brazilwood is known only from Brazil (Cardon
2007, IPCI-Canada 2014), specifically in the coastal
areas of the Mata Atlantica (Fig. 2). Sappanwood
(Caesalpinia sappan) is a shrubby, thorny tree up to
about 8 m in height. Its natural habitat is hilly terrain
with clay soils at low to medium altitudes. The
original range is thought to have included south-
ern China, central India, Burma and Thailand. Over
many centuries, sappanwood was cultivated, then
naturalized over much of northern India, Indonesia,
Papua New Guinea, the Philippines, Solomon
Islands, Sri Lanka, Taiwan and even Hawaii. It is
easily propagated by seeds. Sappanwood today
is used almost exclusively for medicinal purposes
(Cardon 2007).
Nicaragua wood, peachwood or peachum
(Haematoxylum brasiletto) also is known as brazileto,
brasileto (either may be spelled with two ts) or palo de
Brasil, which names occur also in English writings.
All of these names have only exacerbated the con-
fusion described above. Logwood (Haematoxylum
campechianum), the real source of hematoxylin, has
been called campeachy wood, so it is possible that
peachwood was used erroneously in early textile
dyeing literature. The names, brazilette and brsillet,
were used by the French for wood products, admit-
tedly inferior, from Jamaica and other Caribbean
islands, but these almost certainly came from a
related species, Caesalpinia violacea (C. brasiliensis)
with a low content of brazilin (Cardon 2007). Names
aside, Nicaragua wood usually grows only 2  3 m
high as a shrub whose tangled branches are heavily
Brazilin, brazilwood and sappanwood 403

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Fig. 2. Map of Brazil showing the location of the Mata
Atlantia before 1500 (light green) and today (medium
green). Former distribution of brazilwood appears in dark
blue. States containing the Mata Atlantic are labeled, with
those known to contain natural populations of brazilwood
today indicated by an asterisk, as follows: AL, Alagoas*;
BA, Bahia*; CE, Ceara; ES, Esprito Santo*; GO, Goias;
MP, Minas Gerais; PB, Paraiba*; PE, Pernambuco*; PI,
Piaui; PR, Parana; RJ, Rio de Janeiro*; RN, Rio Grande
do Norte*; RS, Rio Grande do Sul; SE, Sergipe*; SP, Sao
Paulo. This figure is a composite of separate maps in IPCI-
Canada (2014), used with permission. Additional
information from Cardon (2007) and Mejia and Buitron
(2008) was added.
thorned. It grows only along the western portion
of Costa Rica and Mexico, and another population
exists in Columbia, northern Venezuela and the
nearby offshore islands. Nicaragua wood is not cul-
tivated (Cardon 2007), probably because of its lower
dye content.
Regardless of origin, the article of commerce was
and is in the form of logs, billets, sticks, chips and
sawdust of heartwood. The outside of larger pieces
is dark red from oxidation of brazilin to brazilein,
while the interior, if reasonably fresh, is nearly col-
orless. The wood has an aroma and bittersweet taste
(Crace-Calvert et al. 1876).
History
Sappanwood
Archeological textiles from Turkistan provide con-
clusive evidence that brazilin had been used since
at least the 1st or 2nd century BC through the 3rd
century AD (Cardon 2007). Sappanwood extract
404 Biotechnic & Histochemistry 2015, 90(6): 401423
apparently was not used originally as a traditional
Chinese dye, because it was not included in a list
from the Western Zhou Dynasty (1045  711 BC),
but it probably was used in China as a medici-
nal during this early period (Ye et al. 2000). It is
mentioned repeatedly in classical texts from India
during the 6  12th centuries AD (Cardon 2007).
Arabic trade records show that sappanwood
arrived in Europe more than 10 centuries ago and
French records date back to 1085 AD (Aguiar and
Pinho 2007). These sources indicate that sappan-
wood came mostly from Japan, the Moluccas (Spice
Islands) and Thailand.
Brazilin from Chinese sappanwood was exported
to Japan during the Asuka period (593  710 AD) to
produce the special color, koro, on silk. In 820 AD,
the Emperor established koro as an official color in
Japanese ceremonial costumes. This was mentioned
subsequently in the Engishiki in 927 AD (Hiroshi
2007), which prescribed its exclusive use by the
aristocracy and Emperor s court. The Engishiki, a
set of 50 scrolls written between 905 and 927 AD,
codified laws, procedures and customs for the Heian
Court. Its influence over Japanese culture remains
to this day, e.g., ceremonial dolls still are dressed in
cloth dyed with brazilin.
Shortly after the Engishiki was published,
European explorers and traders left records of
sappanwood use and cultivation throughout
Southeast Asia. Thereafter, sappanwood became a
highly valued article of commerce. Tax lists from
Ferrara, Italy contain verzino, the Italian word for the
dye, in 1193 and from Barcelona, Spain in 1280 (Leg-
gitt 1944). Three grades of the dye were recognized
in the Commercial Handbook of Pegolotti, a manual
written in 1340 by Florentine banker, Francesco
Balducci Pegolotti, for merchants traveling to
China (Yule 1874). As an aside, Yule translated
the original Italian manuscript, whose title was
variously known as Libro di divisamenti di paesi e
di misuri di mercantanzie e daltre cose bisognevoli di
sapere a mercatanti, [Book of descriptions of countries
and of measures employed in business and of other things
needful to be known by merchants], or more simply
La Practica. According to Pegolotti, the best dye
came from Malabar (southwest coast of India), the
second best from Lambri, Sumatra and the poorest
grade from Siam (Thailand).
Crimson (bluish-) and scarlet (orange-) reds,
produced by the insect-derived dye kermes (kermesic
acid, CI 75460) from the Levant and Eastern Europe,
had already been reserved for high governmental
and ecclesiastical symbols in Europe by the time
Asian brazilin arrived in Europe. Madder (alizarin,
CI 58000) was widely used to dye wool and silk
 shades of dull red or orange, but sappanwood
offered a more robust red color at a cost consider-
ably lower than kermes; thus the dye was adopted
quickly by all, including the lower classes in Europe
(Greenfield 2005).
Sappanwood trade with Asia came to an abrupt
halt shortly after the discovery of the Americas and
subsequent importation of a much richer dyewood
called brazilwood. The change caused consider-
able political and commercial strife among the five
great exploring nations of the 16th century: England,
France, Portugal, Spain and The Netherlands
(Aguiar and Pinho 2007, Auricchio 2014, Defillips
1998, Greenfield 2005, Mohr 1950).
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Brazilwood
Geopolitical aspects
Much of the following historical account, unless
otherwise noted, is derived from Aguiar and
Pinho (2007), an authoritative Portuguese language
on-line pamphlet from the Institute of Botany in
Sao Paolo, Brazil. Defilipps (1998) is another general
historical source. Cabral discovered the northeast
coast of South America in 1500 as he attempted
to sail to India, and he claimed the new land for
Portugal. He landed in what is now Porto Segura
in the state of Bahia. Believing he was on an island,
he named it Ilha de Vera Cruz [Island of the True
Cross].
News of the discovery and its treasure spread
quickly. A map made in Lisbon in 1502 shows
brazilwood trees. By 1503, commerce in the wood
was well established. The dye was very popular
in Europe so the Portuguese Crown declared a
monopoly on brazilwood in 1511 and established an
excise tax of 15%. The tax caused England, France,
Holland and Spain to ignore the monopoly and
to harvest (or pirate) their own timber (Auricchio
2014). France operated out of La France Antarctique,
now Rio de Janeiro. The land was so large that the
Portuguese Crown remained either unaware or
unconcerned until 1530 when two French mer-
chant ships, commandeered by the Portuguese as
they were re-provisioning in Spain, were found to
contain considerable wealth in brazilwood, gold
and parrots. The Crowns solution was to colonize
the land by establishing Captaincies in what was
then called Terra de Santa Cruz [Land of the Holy
Cross]. Captaincies were self-governing administra-
tive regions given to Portuguese nobility and other
friends of the government in the Azores, Madeira
and what later became Brazil. The Brazilian
Captaincies existed from 1534  1549 and effectively
returned control of commerce to Portugal. When
Portugal finally realized the land was part of a con-
tinent, the name was changed to Brasil [Brazil] in
honor of the valuable tree.
Harvesting brazilwood
Once a tree was felled, the bark and sapwood were
stripped off. The wood was so heavy it had to be
cut into billets 5 palmas (hand spans) in length, or
about 110 cm (1.2 yards). These were carried on the
shoulders of local Indians, then later by African
slaves. Eventually ox carts were used as the distance
between the harvest site and a navigable waterway
increased beyond the endurance levels of the carri-
ers. In the early 1600s brazilwood provided more
than half of the annual import revenues for Portu-
gal. When Brazil gained independence in 1822, the
Portuguese monopoly ended, but the country was
so far in debt that it had to use brazilwood to pay
its loans.
Estimates of the yield of brazilwood logs vary
widely. During the first 30 years, 300 metric tons
were shipped annually (Auricchio 2014). Based on
trade records and speculative estimates of average
tree size and density in the forests, 6,000 km2 of
low altitude coastal rainforest (Mata Atlantica) were
devastated during the first century of exploration.
Another estimate claimed 2 million trees of various
species had been cut during that period. During the
16th and 17th centuries combined, more than 91,000
metric tons of brazilwood were harvested; which is
the equivalent of about 527,000 trees.
Secondary market for brazilwood
In 1775, the Frenchman, Franois Xavier Tourte,
fashioned a violin bow of brazilwood. Because of
its superior resonance qualities (described below
under Bow making), demand for the tree grew
even greater. When the market for natural tex-
tile dyes collapsed in the latter quarter of the 19th
century, bow makers became the principal users of
brazilwood.
Environmental aspects of brazilwood
Before the collapse of the natural dye market,
however, the devastation of the Mata Atlantica had
continued almost unabated for nearly 375 years
despite several well-intentioned efforts to the con-
trary, albeit not always for environmental reasons.
The First Royal Charter in 1542 established rules
and punishments for harvesting in an attempt to
protect Portugals economic interests (Auricchio
2014). In 1605, the Kings Phillippe III of Spain and
Phillipe II of Portugal approved another set of rules
Brazilin, brazilwood and sappanwood 405
 to rationalize and regulate the trade in brazilwood.
Harvesters were required to obtain permits from the
landowners. Penalties of death and confiscation of
assets were mandated (Aguiar and Pinho 2007). The
annual harvest was set at 600 metric tons, this time
to limit the supply in Europe so that prices could
be kept artificially high. The Charter Act of 1827
transferred authority to local justices of the peace to
monitor and ban timber cutting. Brazils King Dom
Pedro II in 1850 again prohibited harvesting, but
that too was ignored as agricultural interests caused
forests to be cleared for coffee plantations. None
of these measures had an effect on the decline of
brazilwood. Hoping to avert disaster, Dom Pedros
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daughter, Princess Isabel, limited timber cutting to
a single company, but that was a short-lived mea-
sure. In the 1920s, Presidents Epitacio Pessoa and
Artur Bernandes established a Forest Code and
an accompanying Forest Service, neither of which
solved the problem (Auricchio 2014).
The first effective action, which created the fully
protected Itatiaia National Park, occurred in 1934,
although the remainder of the coastal rain forest
continued to be overexploited. To raise awareness of
the plight of the trees, brazilwood was designated
the national tree and May 3 became Brazilwood Day
in 1972. At that time, the Federal Rural University
of Pernambuco (Universidade Federal Rural de
Pernambuco) developed a program to collect and
plant seeds of brazilwood to prevent extinction of
the species (Auricchio 2014).
A series of regulations and directives finally
provided effective conservation with a goal of
obtaining a sustainable harvest. Regulation 37-N of
April 3, 1992 put C. echinata on the official list of
endangered species (Ministrio do Meio Ambiente,
1992). Decree 3.607 of September 21, 2000 estab-
lished Brazil as a party to the Convention on
International Trade in Endangered Species (CITES)
after establishing acceptable compliance procedures
(Presidencia da Republica da Brasil 2000).
CITES is a treaty ratified by many countries to
regulate trade in endangered species (see CITES
2014a for the text of the Convention). Its goals
are protection and development of endangered
populations to the point where sustainable harvests
are possible. CITES publishes a list of endangered
species, set out in three Appendices (CITES 2014b).
Species in Appendix I are most endangered and
cannot be sold in international commercial trade.
Species in Appendix II are not threatened with
extinction, but require strict regulation of inter-
national trade to prevent further decline of their
populations. Export permits are required and part
of the permit application process requires evidence
406 Biotechnic & Histochemistry 2015, 90(6): 401423
that sustainable harvests are possible. Appendix
III includes species that are not listed in the other
appendices and thus would be unregulated except
that a Party to the Convention, which already
regulates domestic trade, asks for help on an
international scale.
In 2001, a group of bowmakers from 10 coun-
tries became alarmed that brazilwood soon would
be placed in CITES Appendix I, which would make
it unavailable. Their organization, the Interna-
tional Pernambuco Conservation Initiative (IPCI),
developed a partnership with Brazilian authorities
to create a re-forestation project whose ultimate
goal was to establish a sustainable harvest. The
Brazilian Ministry of Agriculture agreed and estab-
lished the Programa Pau-Brasil to begin re-forestation
in the state of Bahia. The international bowmak-
ing community helps fund such projects through
contributions and sale of merchandise. One IPCI
project teamed cacao growers with bowmakers.
Cacao trees fare best when associated with taller
(overstory) trees, and there now are thriving young
plantations using brazilwood to shade the cacao
trees (Rymer 2004). Several Brazilian bowmakers
including Arcos Brazil (http://www.arcosbrasil.
com/), Marco Raposa Bows (http://www.marcora-
poso.com/) and Waterviolet Bows (http://www.
watervioletbows.com.br/) have mounted massive
re-forestation projects of their own, by growing
plants from seed in nurseries and transplanting
them to plantations or natural settings. Because it
takes at least 20  30 years to produce a harvestable
crop, their efforts only now are beginning to pay
off, but their activities definitely were an influential
force in keeping brazilwood off CITES Appendix I.
At the June 2007 meeting of CITES, Brazil won
approval to have brazilwood included in CITES
Appendix II (CITES 2007). This was quickly codified
in Brazil (Ministrio do Meio Ambiente 2008). As
part of that commitment, a comprehensive national
program was created to promote strategic actions
for preserving brazilwood and its natural habitat.
These actions included evaluation of the status of
the species in its disparate locations, identifica-
tion of conservation units and remaining pockets
of Mata Atlantica, and promotion of sustainable
use and commercial farming in the public and
private sectors. The program is linked to state and
local governments as well as civil organizations
(Ministrio do Meio Ambiente 2012) such as Global
Trees Campaign (GTC 2014), another initiative
by bowmakers (IPCI 2014). A measure of success
of these efforts comes from the International Union
for Conservation of Nature (IUCN), whose Red
Book had included brazilwood as an endangered
 species in 2010, but no longer lists it (IUCN 2010,
2014).
To illustrate how the proportion of heartwood
increases with age of the tree, we calculated the
surface area of heartwood and sapwood from trans-
verse cuts across the trunks of 12- and 27-year-old
trees depicted in IPCI-Canada (2014). Heartwood
of the younger trees occupied approximately 19%
of the total cross section, while that from the older
trees measured approximately 56%. Clearly, a sus-
tainable harvest is still one or two decades away,
because of the slow growth of heartwood.
Habitat
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The present status and fate of brazilwood are
closely related to its habitat, the Mata Atlantica
(Fig. 1). This is a rainforest, both coastal and mon-
tane, with extraordinary ecological diversity. It
originally occupied 16% of Brazilian territory, or
1.3 million km2 (one half million miles2). Since
1503, the forests have been cleared for timber
(brazilwood, mahogany, rosewood and cedar) and
agriculture (first sugarcane, then coffee). Estimates
of the current size of the Mata Atlantica range from
7  8% of the original to 11  16%; the higher figure
includes fragments less than 100 hectares ( 0.4
miles2). Because nearly 40% of the current habitat is
fragmented and 80% of the fragments are very small
( 50 hectares or  0.2 miles2), brazilwood restora-
tion efforts face formidable challenges. It is simply
economically and logistically impractical to trans-
port labor, equipment and supplies to innumerable
tiny plots (Aguiar and Pinho 2007, Carvalhal et al.
2014, Mejia and Buitron 2008, Ribeiro et al. 2009).
Today, brazilwood is found in its natural habitat
only in the states of Alagoas, Bahia, Espirito Santo,
Paraiba, Pernambuco, Rio de Janeiro, Rio Grande
do Norte and Sergipe; it had been found in Minas
Gerais until a few decades ago. Figure 2 overlays
the presumed original distribution and current
distribution of Mata Atlantica, together with the
former distribution of brazilwood. Current natural
populations of brazilwood are too small to appear
on the map.
Prospects for Mata Atlantic are bleak in the state
of Minas Gerais, where only 6.8% of the area is
forested (Da Fonseca 1985). In the state of Sergipe,
brazilwood trees were found rarely in 1994; in fact,
only a few small fragments of rainforest survive
there (Rocha 2010). IPCI is working with others to
establish biocorridors to connect these fragmented
ecosystems (Rymer 2004).
The genus Caesalpinia in general and C. echinata
in particular, prefer the drier portions of the Mata
Atlantica, but these areas have changed substantially
during recent geologic history (Cavalcante de Lima
and Werneck da Cunha 2013, Mejia and Buitron
2008). Glacial periods actually were favorable to
this species, because the forests became cooler and
less humid. Under post-glacial conditions, how-
ever, brazilwood became restricted to small refugia.
While Mata Atlantica is widely diverse today in
terms of population structure, climate and elevation,
brazilwood still would be in clumped populations
even without deforestation, because of its limited
potential for dispersal (see next section). Over-
harvesting has simply made remnant populations
smaller. Not surprisingly, three morphologically
and geographically distinct groupings have been
identified as a result of this isolation.
Genetics
Brazilwoods discontinuous distribution had a
profound effect on gene flow. Seed dispersal is
limited; seeds fall to the ground within a few meters
of the parent tree and the pollen:seed flow ratio
of 6.7:1 is very low, i.e., seeds are dispersed very
little and pollen is blown less than seven times
that distance. Clearly, pollen cannot cross the inter-
vening spaces between refugia; therefore genetic
variation within populations is limited (9%), while
inter-population variation is high. Variation among
the three distinct groupings mentioned above
account for 36% of total genetic variation, which
is consistent with the theory that the species has
always lived in clumps with large distances between,
and that the current fragmentation of populations
resulted from re-emergence from refugia caused
by changing sea levels during the Ice Age rather
than by over-harvesting during the past 500 years
(Cardoso et al. 1998, Lira et al. 2003). Re-forestation
efforts must be sensitive to this genetic informa-
tion to avoid massive inbreeding on the one hand
or introduction of trees into areas for which they
are not genetically acclimated on the other. New
genetic markers have since been developed for just
that purpose (Melo et al. 2007).
Components of extracts
Dye content of source wood
Billets of brazilwood typically are deep red on the
outside, but pale yellow in the interior. The yellow
color is due to brazilin and related compounds and
the red is brazilein. Once the heartwood is chipped,
rasped or pulverized into sawdust, brazilin even-
tually is oxidized by air to brazilein. If the dye is
Brazilin, brazilwood and sappanwood 407
 extracted soon after physical reduction of the heart-
wood, it is rich in brazilin with little brazilein, but
in many cases extraction is delayed and the brazil-
ein content rises appreciably. Puchtler et al. (1986)
noted considerable variation in the proportions of
brazilin and brazilein in their samples of dye just
as they did with hematoxylin and hematein in
samples of logwood.
Estimates of dye content of chips or sawdust
are highly variable. Cardon (2007) stated that
brazilwood contains about 1.3% brazilin, but sap-
panwood contains only about 0.05%. By contrast,
Zanin et al. (2012) claimed that sappanwood con-
tained 822% w/w brazilin. The large discrepancy
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could be due to several factors: variation in the
amount of time between pulverizing the wood and
extracting the dye, the age of the tree, whether the
sample contained only heartwood or heartwood
and sapwood, and geographic differences among
trees of the same species. The general consensus
among historical commercial references suggested
that dye contents were as follows: pernambuco 
other brazilwood  sappanwood  peachwood
(Haematoxylum brasiletto)  brazilette (C. violacea).
Other compounds in extracts
Brazilin and protosappannin B (in that order) are
the major components of aqueous and/or alcoholic
extracts of sappanwood (Tong et al. 2013), followed
by protosappannins A, C E and sappanchalcone
(see Washiyama et al. 2009 for structural formulas).
Numerous minor phenolic components also occur,
such as brazilein (Namikoshi et al. 1987, Fu et al.
2008, Zanin et al. 2012). Except for brazilein, these
components probably do not contribute signifi-
cantly to the color of the wood or extract, although
it is possible that some, at least, occur in the natural
synthesis of brazilin (Saitoh et al. 1986) or may
be converted to brazilein during dyeing (Cardon
2007).
Dye chemistry
Molecular structure of brazilin and brazilein
Brazilin (C16H14O5) is a homoisoflavonoid com-
pound (Cardon 2007), whose molecular weight is
286.98 (Lillie 1977). As already noted, it is readily
oxidized by contact with atmospheric oxygen or
other chemical oxidants to brazilein (MW 284.27)
with a loss of two hydrogen atoms to form a
carbonyl. It is in this way similar to hematoxylin
in behavior and structure; the latter possesses an
additional hydroxyl group (Fig. 3).
408 Biotechnic & Histochemistry 2015, 90(6): 401423
Perhaps the earliest reference to the chemical
structure of brazilin (and hematoxylin) is Chevreul
(1808), but William Perkin Jr. and his colleague
Robert Robinson made the definitive determina-
tion. Perkin is best known as the inventor of the
first synthetic dye, mauve (CI 50245), but he also
spent years investigating the structures of brazilin
and hematoxylin. Perkin was recognized in the
1907 inaugural address of the Manchester Library
and Philosophical Society by incoming president
H. B. Dixon for, among other things, his work on
these two natural compounds (Dixon 19071908).
Perkin and several colleagues, notably Robinson,
presented a long series of papers at the Chemical
Society of London and later published them in the
Journal of the Chemical Society Transactions over an 8
year period under the title, Brazilin, haematoxy-
lin, and their derivatives (see citation of Robinson
and Perkin 19011908 for the first eight papers). The
ninth paper in the series (Engels et al. 1908) reca-
pitulated the experiments. Ten years later, Perkins
son, A. G. Perkin and his colleague, A. E. Everest
(1918) presented a thoroughly detailed and more
readable account. Parenthetically, Everest was the
first to isolate and characterize a group of antho-
cyanin dyes (Livingstone 1987). The structure of
brazilein actually was determined by Robinson and
described in another series of papers by Perkin and
Robinson between 1926 and 1928 (not individually
cited here, but summarized by Livingstone 1987).
Morsingh and Robinson (1970) synthesized ()-
brazilin and ()-hematoxylin (formerly called d,
l-stereoisomers), and from those mixtures isolated
()-brazilin and ()-hematoxylin, which were
identical to the natural dyes. Huang et al. (2005)
developed a different synthesis of brazilin that
involved regioselective reagents, but the outcome
again was a mixture of the two enantiomers; the
natural product, ()-brazilin, was not isolated. Yen
et al. (2010) synthesized ()-brazilin, then converted
it to ()-brazilein.
Fig. 3. Chemical structure of hematoxylin (left) and brazilin
(right). Most hydrogen atoms have been omitted.

Fig. 4. Brazilein in three-dimensional conformation to
show asymmetry. Brazilin appears similar.
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A three-dimensional depiction of ()-brazilein
(Fig. 4) shows the molecules left/right, front/back
asymmetry well; the O 11 hydroxyl group projects
forward of the rest of the structure. The original
two aromatic rings are twisted out of a single plane
to accommodate spatially the hydrogen atoms
attached to C 3 and C 14. See Fig. 5 for how atoms
are numbered.
Since Perkin and Robinsons work, the chemical
structures of brazilin and brazilein have been
verified in various ways. The stereochemistry of
both dyes was studied by Craig et al. (1964) and
like hematoxylin and hematein, they are ()-
stereoisomers. NMR characterization of brazilein
was reported by Seon et al. (1997) and Wongsooksin
et al. (2008). Raman and infrared spectra for brazilin
and brazilein were obtained by de Oliveira et al.
(2002), which provided a nondestructive assay for
identifying the dyes in archival and archeological
specimens. Domnech-Carbo et al. (2005) used abra-
sive voltammetry, an electrochemical procedure, to
identify brazilin and hematoxylin from 15th century
Fig. 5. Chemical structures of brazilin (left) and brazilein (right). Non-hydrogen atoms are assigned sequential numbers
by the modeling software. Carbon atoms in solid black circles belong to conjugated systems. Additionally, O21 is a conjugated
atom. Partial atomic charges for numbered atoms are listed in Table 2. Most hydrogen atoms have been omitted.
textiles. Yan et al. (2005) developed a quantitative
method for brazilein using HPLC, with a detection
limit of 0.6 ng/20 l injection volume.
Organic molecules with aromatic systems
(alternating single and double bonds) are con-
sidered to be conjugated, i.e., the electrons of
conjugated atoms do not belong to any one atom,
but are mobile and free to move through the
system. They can be energized by extraneous
forces such as UV or visible light. Light absorbed
by the molecule causes the electrons to go from
a lowest energy ground state to an excited state.
Higher wavelengths of light are absorbed as the
number of electrons in a conjugated system
increases.
Brazilin has two small conjugated systems of six
atoms each (Fig. 5, left). An aliphatic region with a
carbon atom (C 7) and two single bonds separates
the aromatic rings. The system is small, so only low
wavelength (UV) light is absorbed and the molecule
is colorless when pure. With oxidation, the number
of electrons increases to 14 (Fig. 5, right), light is
absorbed at longer wavelengths and the molecule
becomes visibly colored. Compounds with conju-
gated systems of  30 atoms are likely to be fluores-
cent, as brazilin is (Juarranz et al. 1986). Solutions of
brazilin may be colored weakly yellow or yellow-
orange if contaminated with brazilein. Brazilein
solutions are red. Acids cause brazilein to become
orange and alkalis produce a deep crimson color
(Crace-Calvert et al. 1876).
Although brazilin and hematoxylin are closely
related molecules, far more work has been done
on the latter, probably because it has been more
important commercially as a dye since the late
1800s. When oxidized, hematoxylin loses a proton
from one of the paired hydroxyl groups to form a
carbonyl to form hematein. Various investigators
Brazilin, brazilwood and sappanwood 409
 have presented different structural interpretations
(see Puchtler et al. 1986 and Bettinger and Zimmer-
mann 1991a for thorough reviews and their own
proposals); however, neither of these groups of
researchers was able to detect a carbonyl. Molecu-
lar modeling software using quantum mechanical
computations provides a simpler, more comprehen-
sible explanation.
A nominal carbonyl is depicted for brazilein
(Fig. 4), but it is part of a conjugated system.
Unconjugated carbonyl oxygen atoms hold a high
partial charge, e.g., 0.88 in eosin Y, CI 45380,
but the partial charge of brazileins carbonyl
oxygen atom (21 in Table 2) is reduced substan-
tially to 0.25 by its dispersion among 12 carbon
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and c) an anion at O21 and a cation at O12. None of
the structures proposed by either group of research-
ers is demonstrable by quantum mechanical com-
putations. The latter investigators acknowledged
that their structures were not real and that only the
quantum mechanical combination of the structures
describes the actual molecule; using partial charges
to represent the molecule accomplishes a reasonable
description.
Molecular structure of brazalum
Most formulated histological stains derived from
hematoxylin contain a complex of hematein and

aluminum, and thus are known as hemalum solu-

atoms, particularly C6, 16 and 17. Hematein exhibits tions. Likewise, the term brazalum refers to the
the same charge characteristics as brazilein. By corresponding solution of brazilein and alumi-
contrast, Puchtler et al. (1986) chose to depict O21 num. Although several reports have both dyes
as an anion and C7 with a dot to signify a coordina- in their titles, little work actually has been done
tively unsaturated state. Unlike most free radicals, to elucidate the structure of brazalum except for
however, C7 is stabilized by the aliphatic cres- Wongsooksin et al. (2008). Much of the research
cent formed by C19, 8 and 9 (Puchtler et al. 1986). and speculation about the structure of hemalum is
Bettinger and Zimmerman (1991b) proposed three not germane to our review of brazalum, because
resonance structures for hematein: a) a carbonyl at of brazileins more limited opportunity for metal
C16 and O21, b) an anion at O21 and cation at C7, chelation. Hemalum, however, provides a reason-
able starting point for a discussion of brazalum,
although the following is not intended to be
Table 2. Partial charges on non-hydrogen atoms of
a complete description of the many molecular
brazilin and brazilein. species proposed for hemalum. To clarify, we
have standardized the terminology of dye/metal
Partial charges complexes and ratios so that the first term is the
dye and the second is the metal, as in hemalum
Atom Brazilin Brazilein
and brazalum, although many references put the
C1 0.11 0.13 metal first.
C2 0.24 0.20
According to various investigators, hemalum
C3 0.15 0.04
C4 0.13 0.15
consists of 1:1 (Arshid et al. 1954b, Bettinger and
C5 0.12 0.16 Zimmermann 1991b), 2:1 (Arshid et al. 1954b), or
C6 0.18 0.25 1:2 (Harms 1957, Baker 1962, Puchtler et al. 1986,
C7 0.01 0.05 Marshall and Horobin 1973, Marshall et al. 1979)
C8 0.04 0.07 ratios of hematein:aluminum. To some extent, the
C9 0.03 0.01 disagreement has its origin in different experimen-
O10 0.21 0.20 tal conditions. Curiously, none of the work was
O11 0.29 0.30 performed in the acidic pH range that nearly all
O12 0.25 0.24
hemalum stains use. As Bettinger and Zimmermann
C13 0.05 0.04
(1991a) and Puchtler et al. (1986) have shown,
C14 0.15 0.20
C15 0.01 0.01 hematein is neutral or ionic depending upon the pH
C16 0.06 0.29 and this can affect its interaction with aluminum.
C17 0.11 0.22 Unfortunately, after 60 years we still do not know
C18 0.08 0.02 with certainty the molecular structure of hemalum
C19 0.13 0.16 at its conventional staining pH (2.03.5). It is plau-
O20 0.27 0.22 sible, however, that in formulations with nearly
O21 0.25 0.25 equal molar proportions of dye and metal, the 1:1
See Fig. 5 for how the atoms are numbered. Partial charges on
complex is most likely, while in formulations with
three atoms (boldface) are significantly greater in brazilein owing very low proportions of dye to metal, a 1:2 ratio
to charge dispersal from the carbonyl oxygen (O21). should predominate.

410 Biotechnic & Histochemistry 2015, 90(6): 401423

 binding two aluminum atoms, so 1:2 complexes are
impossible.

Physico-chemical properties of brazilin

and brazilein
In recent years, certain structural attributes of
molecules have been used successfully to predict
their behavior as stains, fluorochromes, therapeutic
agents or pesticides; the technique is called quan-
titative structure activity relationships (QSAR).
Several such quantitative measures, taken from
Juarranz et al. (1985), Rashid and Horobin (1991)
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and Dapson (2005) are given in Table 3 for brazilin

Fig. 6. Structure of a 1:1 complex of brazalum. The two
bonds connecting the aluminum atom to brazilein follows
the convention of Rys and Zollinger (1972). Bound water
is not shown.
To our knowledge, the only comparable research
on brazalum is that of Wongsooksin et al. (2008).
These investigators did not use low pH, but they
did vary the molar proportions widely. They
concluded that when there was a two-fold or greater
excess of dye over aluminum, brazalum consists of
a 2:1 complex; otherwise a 1:1 complex prevailed.
Because no common stain formulation has more
dye than aluminum, we conclude that brazalum
staining solutions probably contain 1:1 com-
plexes (Fig. 6). Note that brazilein is incapable of
and brazilein.
Brazilin and brazilein are similar for all para-
meters. Brazilin is lipophilic (positive log P, hydro-
phobic Index  5.0). With a pKa of 5.2, brazilin is
nonionic below pH 3.2 and monoanionic above pH
7.2. Brazilein would be similar.
With five atoms having partial atomic charges
between 0.15 and 0.50, or between  0.15
and  0.30, brazilin and brazilein have moderately
high potential for hydrogen bonding. Two sites in
brazilein (C15O20 and C16O21, Fig. 5) lie adjacent
to one another and thus are likely to form coordina-
tion complexes with metal ions. There is rather low
potential for bonding by van der Waals forces as
evidenced by low values for atoms, d and alpha.
See Dapson (2005) for detailed explanations of how

Table 3. QSAR parameters for brazilin and brazilein.

Parameter Parameter name and significance Brazilin Brazilein

alpha Polarizability (range 69 to  960) 161 201

Measures potential for dipole-induced dipole interactions and
dispersion forces (van der Waals interactions)
CBN, LCF Conjugated bond number, largest conjugated fragment (manual 16 n/a
inspection)
Measures potential for van der Waals interactions
d Dipole moment (range 0 to  100) 1.2 3.5
Measures potential for dipole-dipole and dipole-induced dipole
(van der Waals) interactions
h Hydrogen bonding parameter (range 0 to  11) 5 5
HI Hydrophobic Index (range 1.3 to  8) ( 5.0 hydrophilic,  5.0 7.13 6.49
lipophilic)
Log P Lipophilicity (), hydrophilicity (-)  1.3
MW Molecular weight 286.28 284.26
atoms Number of conjugated () atoms (range 13 to  60) 66 14
Generated by molecular modeling software
Measures potential for van der Waals interactions
pKa pH at which half the molecules are nonionic 5.2 Similar
Z Ionic charge 0 below pH 3.2 Similar
1 above pH 7.2

From Dapson 2005, Juarranz et al. 1985, Rashid and Horobin 1991. n/a  not available.

Brazilin, brazilwood and sappanwood 411

 these parameters are related to various types of
bonding.
Uses of brazilin, brazilein and their
source woods
Biology and histology
The earliest uses of brazilin in biology were sum-
marized by Mohr (1950) and are reproduced here
because we could not access the original papers
cited by Mohr. In 1758, Reichel traced the vessels
of beans (Phaseolus) and lupines (Lupinus) by dip-
ping cut stems into an aqueous extract of brazil-
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wood and concluded from the uptake of dye that
the function of the vessels was to carry sap rather
than air. Similar experiments were carried out
by Hedwig in 1782 and Mayer in 1793. In 1884,
Saefftigen may have been the earliest to use bra-
zilwood extract as a stain for animal tissues (Acan-
thocephalid worms). This was followed in 1889
independently by Breglia in Italy and Flechsig
in Germany using mammalian neurological tis-
sue, but the results were unsatisfactory (Hickson
1901). Both investigators used alcoholic extracts of
brazilwood; Breglia modanted with copper, iron
or lead salts, while Hickson used a lengthy pro-
cedure similar to a Pal-Weigert stain, which had
been developed a few years earlier (Conn et al.
1960). In the 1890s, Rawitz made a brazilin stain
with alum, Heimann used a Delafield-type variant,
Eisen adapted Bhmer s ripened alum hematoxy-
lin to use brazilin/brazilein, and Schaudinn used
his own variant (Mohr 1950). Belling (1928) used
a method based on brazilin/brazilein instead of
carmine (CI 75470) to demonstrate chromosomes
in pollen.
Hickson (1901) provided the most complete
instructions for solution preparation and staining
with brazilein by adapting Heidenhains iron hema-
toxylin procedure. He was the first to claim satis-
factory results for a wide variety of animal tissues
and he noted that brazilin produced polychrome
colors including dark purple chromosomes, brown
cytoplasmic granules and various shades of red for
other components. Brazalum was described by Lee
and Mayer (1907) and thought by Kiernan (1999) to
be more stable than hemalum, because it has one
fewer hydroxyl group and thus is less prone to over-
oxidation.
After the early 1900s, brazilin was not available
reliably, because the source trees became increas-
ingly threatened. Its use in the textile industry
dwindled with the substitution of synthetic dyes.
Dye from sappanwood apparently never entered
412 Biotechnic & Histochemistry 2015, 90(6): 401423
the biological staining market. The only novel use
of brazilin in biological staining since 1900 seems to
have been by Lillie (1956), who found that it behaved
similarly to hematoxylin and other catechols in a
Meirowsky reaction for keratohyalin. This reaction
does not need a mordant to stain the granules black.
Lillie (1965) reported that red color was produced
with aluminum and tin mordants, red-brown with
copper, and a mixture of blue-green and red-brown
with iron.
Lillie et al. (1976) tried 26 dyes, including bra-
zilin, as substitutes for hematoxylin, but surpris-
ingly, brazilin was considered unsatisfactory for
staining nuclei. Puchtler et al. (1986) found that
their samples of brazilin did not produce suitable
brazalum stains when oxidized with sodium iodate,
but did perform satisfactorily without oxidation,
which suggests that most of the dye had already
been oxidized to brazilein by atmospheric oxygen.
These investigators also found that brazilin did not
work as a hematoxylin substitute in Weigerts or
Heidenhains iron formulations, Verhoeff s elastin
stain or a phosphotungstic acid brazilin stain for
striated muscle. Given the findings by Puchtler et al.
(1986) that both dye content and the ratio of brazilin
to brazilein were highly variable, the frequent fail-
ure of brazilin/brazilein to stain nuclei selectively
is not surprising.
Brazilin is listed as a fluorochrome by Rost (1995)
and it has been used as a fluorescent probe for mito-
chondria in living cells (Rashid and Horobin 1991).
The mechanism is ion trapping, which involves
interplay between the dyes pKa (5.2) and the vari-
ous pH environments within the cell. Between pH
3.2 and 7.2, some portion of the dyes molecules are
nonionic and thus lipophilic; therefore, the dye can
cross cell and organelle membranes. Once within
mitochondria, however, most dye molecules become
anionic, because the internal pH of the organelle
when metabolically active is slightly alkaline; thus
the brazilin anions are trapped within. Dye mol-
ecules that diffuse through the rest of the cell soon
move out, which leaves mitochondria quite visible
under UV light.
Small amounts of brazilin were sold to fiber
artists in the latter quarter of the 20th century as
craft dyers rediscovered natural dyes. These prod-
ucts were too impure for histological use and
likely came from one of the lesser sources (Caesal-
pinia violacea, Haematoxylon brasiletto or Caesalpinia
sappan). One of us (RWD) obtained a large quan-
tity of high quality material and produced a com-
mercial Harris-type brazalum stain trade-named
Brazilliant (Anatech Ltd., Battle Creek, MI) in
2001 (Fig. 7, Anon. 2002).

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Fig. 7. A Harris-type brazalum stain with Alcian blue.
Nuclei are dark red, cytoplasm pink, goblet cells blue.
Textile dyeing
In the textile dyeing industry, there are two classes
of red dye woods, insoluble and soluble. The for-
mer, including sandalwood, barwood and cam-
wood, produce a complex mixture of resinous red
compounds (CI natural red 22) somewhat related to
some of the constituents of crude extracts of brazil-
wood or sappanwood, but more resistant to fading
by light because of their high tannin content (Car-
don 2007). Soluble red dye woods contain brazilin.
Dye binding
As discussed above, textile fibers or whole garments
have been colored with derivatives of brazilin for at
least several thousand years. Because pure brazilin is
colorless, brazilein must be the coloring agent. Wool
and nylon usually are dyed with brazilein from an
acid bath to expand the fibers and to keep the dye
in a non-ionized state. According to Arshid et al.
(1954a) the mechanism of attachment to wool is by
hydrogen and ionic bonding, but to cellulose (cot-
ton, linen etc.) it must be by van der Waals forces.
Their reasoning was that brazilein (and hematein)
could not compete with hydrogen-bonded water
molecules attached to the hydroxyl groups of cel-
lulose. Why these dyes could hydrogen bond to
proteins and not cellulose was not addressed. The
ability of brazilein to bind by van der Waals forces
is minimal (see Physico-chemical properties and
Table 2 above).
Colors
The typical color of fabric dyed with extracts of brazil-
wood or sappanwood without mordant varies from
pink to red; clearly, brazilein is the agent responsible.
Mordants expand the color range from brown with
chromium to purple-black with iron (Crace-Calvert
et al. 1876). Copper gives a muddy light brown
with hints of purple and green, aluminum produces
orange-red, while tin gives the most vibrant red
(Fig. 8). The pH of the dyebath also is important,
more orange hues are produced in acidic solutions.
If the fabric or yarn is treated with an alumi-
num, cobalt, chromium or iron salt before dyeing,
brazilein forms a metal coordination complex in situ
(Arshid et al. 1954b). In earlier days, the dye usually
was applied to the cloth first, followed by the addi-
tion of mordant to the dye bath (Bemiss 1815), but
the reverse sequence retains more dye in the fibers.
Bemiss (1815) used brazilwood with potassium
aluminum sulfate in a copper kettle to obtain red,
or varied the shade by adding potassium carbonate
to achieve crimson, urea from aged urine to obtain
maroon, or tin (stannous) nitrate to obtain claret.
In various recipes, the mordant was applied either
before or after the dye. Bemiss (1815) frequently used
madder (CI natural red 8 that contained alizarin,
CI 75330, and its relatives) with brazilwood either
mixing the two dyes in the same bath or by dye-
ing sequentially, but he preferred brazilwood alone
for a purer red color. No doubt the less expensive
and more widely available madder was used for
economic reasons. Bemiss (1815) also was mindful
of expense in another recipe that involved both
brazilwood and logwood and suggested substitu-
tion of peachwood for brazilwood with no adjust-
Fig. 8. Skeins of wool yarn dyed with brazilwood dust and
four different mordants. From left to right: tin, copper, iron
and alum.
Brazilin, brazilwood and sappanwood 413
 ment for dye content, although he knew full well
that peachwood was inferior. To his credit, Bemiss
did state, this [the substitution] I leave to your own
choice (Bemiss 1815).
Fastness
Historically, fabric dyed with brazilin/brazilein
was known to fade upon exposure to sunlight (see
Puchtler et al. 1989 for information and references
spanning the years 13831670). It is not known
whether mordants were used in these examples.
Later dyers developed processes that substantially
increased the fastness of dyed and printed fabrics,
e.g., Crace-Calvert et al. (1876) used sumac extract,
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which is rich in tannins (Veitch et al. 1920), on
fabric dyed with extract of sappanwood. Mordants
of aluminum, chromium and iron also increased
fastness to light.
Ye et al. (2000) studied fading of alum-mordanted
brazilein on silk in the absence of light. Appreciable
fading was evident after 22 weeks of exposure to
400 ppb ozone, a concentration similar to highly
polluted urban air. This is important for conser-
vators of silk artifacts, silk textiles, silk paintings
and silk tapestries that might be stored in urban
environments. It should be noted that with many
dyes, silk fabric is unusually sensitive to sunlight
or daylight-type fluorescent fixtures.
Traditional folk medicine
Traditional medical practices throughout Asia,
especially in China, India, Indonesia, Japan and
South Korea, use sappanwood and its extracts for
a variety of treatments as described in two recent
comprehensive reviews (Badami et al. 2004, Zanin
et al. 2012). Different cultures use the medicaments
for different conditions and diseases. Reported uses
include promoting menstruation, blood flow and
diuresis; reducing pain and swelling; and treating
skin diseases, high blood sugar, microbial and viral
infections, cancer and atherosclerosis. In China,
brazilin is used as an analgesic and as therapy for
thrombosis and tumors (Soka 1985). In Indone-
sia, a decoction of sappanwood is used to lighten
skin color (Chun 2002), which is curious in light
of Lillies (1956) use of brazilin in the Meirowsky
reaction to blacken keratohyalin.
Scientific research into the pharmacological
properties of purified brazilin began in the late
1970s. In many cases, these studies used whole
extracts containing inter alia, flavonoids, phenols,
protosappanins, quinones, steroids and terpenes
(Hikino et al. 1977, Zanin et al. 2012). More recently,
414 Biotechnic & Histochemistry 2015, 90(6): 401423
however, an increasing number of investigators
have isolated and tested specific components; those
related to brazilin and brazilein specifically are
reviewed in the following section.
Curiously, there is little information about
brazilin-containing woods in Brazilian ethno-
pharmacology. Zanins review (Zanin et al. 2012) is
authored by Brazilians and it mentions Caesalpinia
echinata, but there is no record of its use in local folk
medicine. Brazilwood is not included in an exten-
sive summary of pharmacologically interesting
plants produced by Brazils South American Office
for Anti-cancer Drug Development (Mans et al.
2000). Defilipps (1998) does state that brazilwood
is used in tonics and in rinses to soothe toothaches.
By contrast, the Digital Library of Traditional Mexi-
can Medicine (Anon. 2009) includes palo de brasil
(Haematoxylum brasiletto) as a remedy for diabetes,
dizziness, fainting, gastrointestinal distress, high
blood pressure, inflammation and kidney ailments.
Wood chips of the species are offered for sale as
a Cuban folk curative to lower blood sugar and
cholesterol (De Gezelle 2004).
Pharmacological properties
Some components of wood extracts other than
brazilin or brazilein also have pharmacologi-
cal properties (Liu et al. 2009, Washiyama et al.
2009). Consequently, reports of activity of unpuri-
fied extracts of source trees must be viewed with
caution when considering attributes of the two
dyes discussed here. For a thorough review of the
pharmacologic properties of extracts from many
species of the genus Caesalpinia, see Zanin et al.
(2012). The following is an account of the specific
effects of brazilin or brazilein unless otherwise
noted.
Antibacterial
Pratt and Yuzuriha (1959) applied aqueous
extracts of Haematoxylum brasiletto heartwood and
noted bactericidal effects on Salmonella typhi and
Micrococcus pyroxenes, and bacteriostatic effects on
E. coli. They theorized that brazilin and/or brazilein
were responsible, although no attempt was made to
isolate the components. Nevertheless, later research
by others (below) appears to support their claim.
MRSA refers to methicillin-resistant Staphy-
lococcus aureus, but resistance can be toward any
beta-lactam antibiotics (the penicillins and cepha-
losporins). Other bacterial species also have
become resistant to drugs of choice, e.g., vancomy-
cin-resistant enterococci (VRE). Brazilin is a potent
antibiotic that kills MRSA and VRE by inhibiting
 incorporation of thymidine into DNA and serine
into proteins. Culturing bacteria at sublethal doses
of the dye for 20 generations did not lead to devel-
opment of resistance (Xu and Lee 2004).
Antihelminthic
Brazilein is significantly toxic to Hymenolepis nana,
the most common cause of cestode infections
globally, when administered to isolated parasites.
It was less effective against Anisakis simplex, the
nematode commonly ingested when eating raw,
lightly pickled or salted sea food including sushi,
sashimi and Scandinavian raw fish (Liang et al.
2013).
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Antiobesity
Brazilein inhibits accumulation of lipid droplets
in adipocytes. It also inhibits the regulatory pro-
cess that involves transcription factors that govern
adipogenesis (Liang et al. 2013).
Antioxidant
Brazilin is an antioxidant under certain circum-
stances. Bromotrichloromethane causes oxidative
damage by free radicals including increased lipid
oxidation and cell membrane leakage in cultured
rat hepatocytes. These effects are inhibited by the
dye (Moon et al. 1992). Under more natural con-
ditions, the enzyme, heme oxygenase-1 (HO-1),
protects cells against oxidative injury from sun-
light, free radicals and certain chemicals. Purified
Table 4. Examples of antitumor activity by brazilin and brazilein.
Compound Cell lines
Brazilin Bladder T24*
Glioblastoma*
Ovarian
Brazilein Basal cell carcinoma BCC
Epidermoid carcinoma A431
Leukemic monocyte macrophage RAW264.7
Malignant melanoma
Premalignant keratinocytes HaCaT*
Squamous cell carcinoma SCC25
Liver Hep 3B*
Liver Hep62*
Breast MDA-MB-231*
Breast MCF-7*
Lung A549*
Gingiva Ca922*
Cell lines marked with (*) are human. Actions include: (1) inhibits proliferation, (2) inhibits apoptosis, and
(3) cytotoxic.
brazilin causes increased activity of HO-1 (Choi
et al. 2007) in part by disrupting several intracel-
lular and extracellular pathways that activate HO-1
(Choi and Kim 2008).
In pharmacological research, the activity of
prospective antioxidants is compared with the
vitamin E analog, Trolox, a standard antioxidant.
Brazilein compared favorably in scavenging radical
cations with a score of 0.98 g/1.0 g Trolox (Liang
et al. 2013). Antioxidant activity also can be mea-
sured by assessing the inhibition of lipid oxidation
of membranes. In this case, brazilein was more
effective than the antioxidants Trolox and rutin
(Liang et al. 2013). Brazilin showed less than half
the antioxidant activity of vitamin E in the linoleic
acid emulsion test (Sasaki et al. 2007).
Inflammation is caused in part by the production
of superoxide anions. Brazilin, brazilein and a vari-
ety of synthetic analogs showed potent inhibitory
effects on superoxide generation (Yen et al. 2010).
Antitumor
The antitumor activity of brazilin has been rec-
ognized for centuries in Chinese herbal medi-
cine. Several recent studies have validated this
claim under experimental conditions (Table 4).
Interestingly, hematoxylin has similar antitumor
properties by inducing apoptosis and/or direct
affects on T24 human urinary bladder cancer cells
(Ren 2008); it also prolonged survival of mice
with experimentally induced hepatitis hepatomas
(Ren 2009).
Action References
1 Ren et al. 2011
1 Lee et al. 2013
1, 2 Zhang et al. 2010a,b
1 Liang et al. 2013
1 Liang et al. 2013
Liang et al. 2013
1 Liang et al. 2013
1 Liang et al. 2013
1 Liang et al. 2013
3 Yen et al. 2010
3 Yen et al. 2010
3 Yen et al. 2010
3 Yen et al. 2010
3 Yen et al. 2010
3 Yen et al. 2010
Brazilin, brazilwood and sappanwood 415
 Binding to DNA
Some chemotherapeutic and antibacterial com-
pounds work by binding to DNA. Bhakta et al.
(2013) showed that spectral properties of brazilin,
as measured by ultraviolet, visible, infrared and
circular dichroism spectroscopy, shifted over a 3
h period upon the addition of DNA, which sug-
gests that the dye binds to DNA. Further, comput-
erized molecular docking simulations indicated
that brazilin could bind readily to DNA; the
conclusions are problematic, however, because
unoxidized dye was used without a mordant. As
indicated previously under Mechanism of stain-
ing, brazilin itself does not stain (bind) to nuclei
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unless it has been oxidized and complexed with a
metal ion such as aluminum or iron. Bhakta et al.
(2013) actually may have used brazilein, not bra-
zilin, because they extracted the dye from sap-
panwood using the method of Fu et al. (2008),
who isolated brazilein from sappanwood chips.
Nevertheless, a mordant was lacking, which
leaves the mechanism of binding in question.
Clearly further work is required to substantiate
these findings, because they could be significant
for DNA-targeted therapeutics.
Blood clotting
Brazilin inhibits aggregation of rat platelets in
vitro by decreasing phospholipase activity and
increasing intracellular free calcium ions (Hwang
et al. 1998), which gives credence to the use of the
dye as an anti-thrombocytic agent in traditional
Asian folk medicine. Brazilin also appears to facili-
tate clotting, however. In damaged human blood
vessels, clotting begins when platelets encounter
exposed subendothelial collagen. Affinity and
efficacy of collagen receptors on the platelets are
enhanced by brazilin (Chang et al. 2013). These
investigators attributed the conflicting results to
species differences. More likely, the two groups of
researchers were viewing the complex process of
clotting from different perspectives: inhibition of
clotting in normal vessels vs. clotting induced by
damaged vessels.
Cardiac effects
Inotropes are chemicals that alter the force of car-
diac muscular contractions, either strengthening
(positive effects) or weakening (negative effects)
contractions. Brazilein acts as a positive inotrope by
inhibiting Na()- and K()-ATPase, and therefore
might be used to enhance cardiac function in certain
cardiomyopathies (Zhao et al. 2006)
416 Biotechnic & Histochemistry 2015, 90(6): 401423
Hypoglycemic effects and diabetes
Brazilin lowers blood glucose levels by several
physiological routes. Glucose normally is taken
up into muscle and fat cells under the influence of
insulin in a multi-step pathway that culminates in
transport across the cell membrane by the protein,
GLUT4 (glucose transporter type 4). Brazilin may
increase glucose transport by moving GLUT4 from
intracellular vesicles to the cell membrane (Khil
et al. 1997, 1999). In addition to increasing glucose
transport, brazilin also influences enzymatic path-
ways that involve glucose metabolism, which
causes increased glucose oxidation, glucose-6 phos-
phate dehydrogenase activity, glycogen synthesis
and glycolysis. (Moon et al. 1990, 1993). Brazilin
also decreases gluconeogenesis in hepatocytes that
have been stimulated with glucagon or fatty acids
(Won et al. 2004). Brazilin elevates levels of fruc-
tose-2,6-bisphosphate, a regulator of gluconeogen-
esis (You et al. 2005) and enhances insulin receptor
function (Kim et al. 1995, 1998). When normal and
induced-diabetic rats were fed Sappan Lignum
extract, both brazilin and protosappanin B were
retained longer in the induced diabetic group, which
suggests that these compounds are accumulated in
diabetic individuals (Tong et al. 2013).
The enzyme that initiates cataract formation in
diabetic individuals is aldose reductase. Many fla-
vonoids inhibit lens aldose reductase (Varma and
Kinoshita 1976) including brazilin and hematoxylin
(Moon et al. 1985).
Inflammation and immune functions
Brazilin can affect immune functions in several
ways including mitigation of asthma, inhibition of
complement fixation, lessening of general inflam-
mation and prevention of immunologic tolerance.
Two experimentally induced diseases were also
affected positively. Table 5 lists how all of these
events were accomplished.
Neurological disorders
g-Aminobutyric acid (GABA) is a neurotransmitter
that is deactivated in a complex system (GABA
shunt regulating enzymes) that includes succinic
semialdehyde reductase (SSAR). Abnormally low
levels of GABA, possibly owing to an excess of
SSAR, may be associated with some neurologi-
cal disorders such as Parkinsonism, epilepsy and
convulsions. Brazilin produces dose-dependent
inactivation of SSAR and has been proposed for use
in anticonvulsant therapy (Baek et al. 2000).
Ischemia following stroke or brain trauma
causes activation of endothelial cells, which then
 Table 5. Examples of how brazilin affects inflammatory and immune responses.

Condition Action of brazilin References

Asthma
Complement fixation
General inflammation
Halothane-induced hepatitis
Immunologic tolerance
Infections in mice with induced
type I diabetes
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Decreases interleukin-4 and -5, which inhibits T Lee et al. 2012
helper cell activity
Anti-complementary effects (inhibits lysis, Oh et al. 1998
phagocytosis and anaphylaxis)
Activation of heme oxygenase-1; suppression of Hu et al. 2009,
iNOS mRNA Sasaki et al. 2009,
Bae et al. 2005
Prevents proliferation of splenocytes and Choi et al. 1997
prevents inhibition of suppressor T cells
Inhibits suppressor cell activity Mok et al. 1998
Restores normal cellular immune responses Yang et al. 2000

produce reactive oxygen species. This in turn leads
to an inflammatory response when blood flow is
restored (ischemic re-perfusion). Brazilein admin-
istered after re-perfusion reduces the inflamma-
tion. The mechanism involves decreased mRNA
levels of the pro-inflammatory cytokines, tumor
necrosis factor-alpha (TNF-alpha), interleukin-6
(IL-6) and nitric oxide synthetase (iNOS) (Shen
et al. 2007).
Skin care
Acne vulgaris is associated with hyperkeratinization
and increased production of sebum by sebaceous
glands, and it usually is accompanied by the bacte-
rium, Propionibacterium acnes. The organism releases
lipases that degrade sebum to free fatty acids that in
turn induce inflammation. Inflammation generates
free radicals, so the body suffers oxidative stress as
antioxidants are depleted. An effective therapeutic
for acne, therefore, should be antibacterial toward
P. acnes, inhibit lipase activity and possess anti-
oxidative properties (Batubara et al. 2009). Brazilin
purified from sappanwood had sufficient potency
in all three categories to suggest its use as an anti-
acne treatment (Batubara et al. 2010).
UV-B radiation causes loss of viability of der-
mal fibroblasts, generates reactive oxygen species
and activates the collagenase matrix metalloprotei-
nase (MMP) that breaks down interstitial collagen.
Brazilin affords protection against these events (Lee
et al. 2012).
Vasodilation
Brazilin causes vasodilation by increasing the influx
of extracellular calcium ions into endothelial cells,
which in turn activates a calcium ion/calmodulin-
dependent synthesis of nitric oxide. Nitric oxide is
transferred to smooth muscle cells, which activates
another cascade system that results in relaxation of
muscle tension (Xie et al. 2000, Hu et al. 2003).
Manufacture of musical instruments
The modern violin bow was developed between
1785 and 1790 by Francois Xavier Tourte in France.
Prior to that, bows were crafted of dense European
or tropical hardwoods. Each was cut with a length-
wise camber (arch). Camber is critical for sound
quality (Ablitzer and Dalmont 2011). Among other
changes, Tourte used pernambuco heartwood from
Brazil, previously valued solely for its dye. Brazil-
wood has several outstanding qualities for bows.
When heated, it can be bent into a curve that remains
fixed after cooling, so that the wood does not need
to be milled to achieve a camber. It is very dense,
flexible and beautiful (ICPI-Canada 2014).
The secret of a good violin (or other stringed
instrument) bow is its ability to preserve the vibra-
tions created when the bow is drawn across the
strings. Vibrations are energy and that energy is
dissipated over time (the sound diminishes and/
or the pitch changes); a good bow retards the dimi-
nution of energy. This quality is measurable and is
expressed as the loss tangent or loss factor. A low
loss factor is desirable. Pernambuco, the brazilwood
with the highest content of brazilin and related
compounds, has a low loss factor; less desirable
wood has a high loss factor. When an extract of
pernambuco that contained protosappanin B and
brazilin was impregnated into spruce wood, how-
ever, the loss factor decreased significantly. The
phenomenon was thought to involve hydrogen
bonding. The investigators suggested that the dye
and its relatives are responsible for pernamubos
musical attributes (Matsunaga et al. 2000). Why

Brazilin, brazilwood and sappanwood 417

 Fig. 9. Brazilwood pegs in a lute.

Tourte chose pernambuco is not known and it is

doubtful that he understood the physics of loss
tangents, but he certainly made a sound decision.
Virtually all professional bows since have been
made of pernambuco.
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Unrelated to bows, but in the realm of stringed

instruments, brazilwood also has been used for the
pegs in vihuelas. The instrument shown in Fig. 9
is a modern reconstruction of one of three surviv-
ing Medieval lute-like vihuelas. The instrument
shown is based on Vihuela in a (E0748), part of the
museum collection of the Cit de la Musique, Paris,
France. See http://www.lutesandguitars.co.uk/
htm/cat12.htm for more detailed information,
especially under the section, Models currently
available, 4. Vihuela in a.

Miscellaneous applications
Before the Portuguese arrived in South America,
local inhabitants used brazilwood for making
bows and arrows, and they used the dye for col-
oring ornaments including feathers (Auricchio
2014). Since the 1500s, brazilwood has been used
for cabinetry and even today can be found in Brazil
in such objects as ornate household doors (Fig. 10).
Heartwood extract from Caesalpinia sappan is used in
several Asiatic countries today to enhance the color
of wine and other beverages, and meat (Badami
et al. 2004). The component responsible undoubt- Fig. 10. An ornate door made of brazilwood. Picture taken
in 2014 in a home in Sao Paulo, Brazil.
edly is brazilein. Finally, in Brazil, C. echinata is a
popular shade and landscape tree, although in
colder regions it does not flourish.
Declaration of interest: The authors report no
conflicts of interest. The authors alone are respon-
Acknowledgments sible for the content and writing of this paper.

Chad Fagan, Richard Horobin and John Kiernan

graciously obtained copies of references we were
unable to locate. Sue Payne of Conner Prairie
Interactive History Park, Fishers, IN, shared her
knowledge of dyeing with natural dyes and allowed
us to photograph her samples of brazilwood-dyed
yarn. RWD is particularly grateful to co-author CLB
for translating all of the Portuguese documents.
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