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Pilocarpine and related alkaloids in Pilocarpus Vahl (Rutaceae)

Article · January 2011

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In: Alkaloids: Properties, Applications… ISBN: 978-1-61668-974-2
Editor: N. M. Cassiano, pp. 63-80 © 2010 Nova Science Publishers, Inc.

Chapter 3

PILOCARPINE AND RELATED ALKALOIDS IN

PILOCARPUS VAHL (RUTACEAE)

Alexandra C. H. F. Sawaya1,, Ilka N. Abreu1,2, Nathalia

L. Andreazza1, Marcos N. Eberlin3, Paulo Mazzafera1,*
1
Plant Biology Department, Institute of Biology, State University of
Campinas, UNICAMP, CP 6109, Campinas, 13083-970, SP, Brazil
2
Scottish Crop Research Institute, Department of Plant Products and Food
Quality, Invergowrie, Dundee, DD2 5DA, Scotland, UK
3
Thomson Mass Spectrometry Laboratory, Institute of Chemistry, State
University of Campinas, UNICAMP, Campinas, 13083-970, SP, Brazil

ABSTRACT
Pilocarpine is mainly known as a drug for the treatment of glaucoma
and it is also used as a stimulant of sweat and lachrymal glands. Species
of the genus Pilocarpus are collectively named jaborandi in Brazil and
their leaves are the only known source of this imidazole alkaloid.
Pilocarpine is mainly obtained from two species, Pilocarpus microphyllus
and Pilocarpus jaborandi and, despite the economical and
pharmacological importance of this alkaloid, very little is known about
pilocarpine, from basic information on the contents in different jaborandi
species and plant tissues to the biosynthetic route and the metabolic

*
Corresponding author: pmazza@unicamp.br
64 Alexandra C. H. F. Sawaya, Ilka N. Abreu, Nathalia L. Andreazza et al.

control. This review will focus briefly on the genus jaborandi, then on
what is known about pilocarpine biosynthesis followed by possible
biotechnological applications aiming to produce the alkaloid in vitro.
Finally, the alkaloids found in this genus, their plant sources and
pharmacological applications will be reviewed.

Keywords: Biosynthesis; genetic variation; imidazole alkaloid; Jaborandi

1. PILOCARPUS, THE SOURCE OF PILOCARPINE

Pilocarpus Vahl (Rutaceae) is a neotropical genus comprising shrubs and
trees, with species distributed from the south of Mexico, throughout Central
America and the Antilles, as far as the lower latitudes of South America
(Kaastra, 1982; Skorupa, 1996). The name of the genus is probably based on
the shape of the mericarps, as pilos means felt hat in Greek and carpos means
fruit (Kaastra, 1982). The genus has 17 species and 14 of them are found in the
Brazilian territory, with the majority in the oriental part of the country
identified as the genetic diversity center of the species (Oliveira, 2007).
Several species of Pilocarpus are popularly known in Brazil as jaborandi,
which comes from the name of these plants in the Tupi-Guaraní language (ya-
mbor-endi) meaning ―the one who causes mouth dripping (Holmstead et al.,
1979).
Pilocarpus is the only source of pilocarpine, an imidazole alkaloid with
pharmacological activity, used in eye-drops for the treatment of glaucoma, and
also for the stimulation of sweat and lachrymal glands (Goodman and Gilman,
2001; Valdez et al., 1993). Other imidazole alkaloids were isolated from
jaborandi but little is known about their pharmacological properties (Abreu et
al., 2007a; Abreu et al., 2007b; Andrade-Neto et al., 1996; Kaastra, 1982; Link
and Bernauer, 1972; Santos and Moreno, 2004; Sawaya et al., 2008).
Apparently, all Pilocarpus species have pilocarpine but in varied
concentrations (Joseph, 1967; Sawaya et al., 2010; Sousa et al., 1991;
Voigtlander et al., 1978). However, only two species: Pilocarpus microphyllus
Stapf ex Holmes and Pilocarpus jaborandi Holmes have economical
importance as they are known to have a high pilocarpine concentration in the
leaves and also because they have a broad geographical distribution (Joseph,
1967; Kaastra, 1982; Sousa et al., 1991). The distribution is an important
aspect, as the leaves used for pilocarpine extraction were (until some years
ago) exclusively obtained from native plants growing in the wild and the
 Pilocarpine and Related Alkaloids in Pilocarpus Vahl (Rutaceae) 65

intensive collection made some Pilocarpus species to be included in a list of

endangered Brazilian plants (IBAMA, 1992). The Brazilian state of Maranhão
was the main producer of jaborandi leaves and P. jaborandi, P. microphyllus,
P. alatus and P. trachylophus were the species in the endangered list
(Pinheiro, 1997, 2002). The leaves were harvested by people living in or near
the forest and the pharmaceutical company Merck was the only buyer.
Repeated leaf collection induced plant death, vigor and plant height reduction,
as well as decrease in leaf size (Pinheiro, 1997). Curiously, from a
phylogenetic point of view the above mentioned four species form a unique
clade and their phylogenetic relationships are in a certain degree associated
with their geographical distribution (Oliveira, 2007).
The uncontrolled exploitation accelerated actions for domestication of
native jaborandi species, an initiative by Merck (SUDEMA, 1970), which was
undertaken in the Maranhão state starting in 1969 (Vieira, 1999). A notable
annual mark of 4,000 kg of leaves/ha was reached in the Merck farm but in
recent years the production is about 1,400 Kg of leaves/ha. In 2002, the
Centroflora Group (Vegeflora Extrações do Nordeste Ltda -
www.centroflora.com.br) became responsible for the pilocarpine extraction
from the jaborandi leaves but Merck still carries out the purification and trades
the alkaloid.

1.1. Biosynthesis of Pilocarpine

It has long been suggested (Cordell, 1981) that pilocarpine and other
analogous imidazole alkaloids in plants are derived from histidine but no proof
was given. Dewick (1997) also indicated that pilocarpine is probably derived
from histidine and additional carbon atoms would come from acetate and
threonine, but again without any experimental confirmation. Both suggestions
come from the similarity of this amino acid structure and the imidazole ring
(Fig. 1). However, this may not be a rule as the imidazole alkaloid anosmine is
originated from two lysine molecules (Hemscheidt and Spenser, 1991).
(3-3H-Threonine and (3-3H)histidine were used to study the biosynthesis
of pilocarpine in calli obtained from leaf peduncle (Abreu and Mazzafera,
unpublished results). After 12, 24 and 48 h of incubation pilocarpine was
extracted (Avancini et al., 2003) and analysed in HPLC coupled to a UV and
radioactivity detectors (pumping a scintillation cocktail). Radioactivity was
detected in the pilocarpine peak in all analyzed incubation periods and with
66 Alexandra C. H. F. Sawaya, Ilka N. Abreu, Nathalia L. Andreazza et al.

both labeled amino acids, suggesting indeed that both amino acids may be
involved in the biosynthesis of pilocarpine. However the radioactivity
incorporation was very low, varying from 0.02 to 0.06%. Labeled amino acids
(14C-histidine, 14C-arginine and 14C-ornitine) were also used to study the
biosynthesis route of the imidazole alkaloid stevensine in Teichaxinella
morchella and about 0.2% incorporation was observed (Andrade et al., 1999).
Such low radioactivity incorporation in the target alkaloid might be due to the
destination of part of the labeled amino acids to the synthesis of proteins and
other amino acid derived compounds. Indeed, in our analysis of the jaborandi
extracts in HPLC described above, several other peaks of radioactivity were
identified (Abreu and Mazzafera, unpublished results).

L-Theronine
OH O

OH
O
NH2
CH3 R
N
OH + N
OR O
NH2
N
O
N
L-Histidine O
Pilocarpine
C CoA

CH3
Acetyl-CoA

Figure 1. Probable biosynthesis of pilocarpine from histidine and additional C atoms

from acetyl-CoA and threonine (Adapted from Dewick, 1997).

Interested in finding a model to study the biosynthesis route of pilocarpine

in jaborandi, different approaches to increase the alkaloid content in seedlings
have been tested (Avancini et al., 2003) and then in calli (Abreu et al., 2005)
or cell suspension cultures (Abreu et al., 2007b; Andreazza et al., 2008). When
calli were half-immersed in a liquid medium containing 0.05, 0.15, and 0.75
mM of threonine or histidine there was a significant increase of the alkaloid
content (medium + cells) at the highest amino acid concentration, supporting
the hypothesis that both amino acids may participate in or act as precursors of
pilocarpine biosynthesis in jaborandi (Abreu et al., 2005).
A comparison of the mass spectrometry fingerprint [ESI (+) - MS] of
alkaloid extracts from leaves and cell suspension cultures of P. microphyllus
identified 3-nor-8(11)-dihydropilocarpine, pilocarpine, anhydropilosine,
 Pilocarpine and Related Alkaloids in Pilocarpus Vahl (Rutaceae) 67

pilosine and three new alkaloids (Abreu et al., 2007a). A further and more
detailed study (Abreu et al., 2007b) on the alkaloid profile of P. microphyllus
in different seasons and parts of the plant by electrospray ionization mass
spectrometry fingerprinting allowed the identification of these and other new
alkaloids: (1) pilocarpine, (2) pilosine, (3) 3-anhydropilosine, (4) 13-nor-
8(11)-dihydropilocarpine, (5) 3-(3-methyl-3H-imidazol-4-ylmethyl)-1-phenyl-
but-3-en-1-one, (6) 3-hydroxymethyl-4-(3-methyl-3H-imidazol-4-yl)-1-
phenyl-butan-1-one, (7) 3-benzoyl-4-(3-methyl-3H-imidazol-4-ylmethyl)-
dihydro-furan-2-one and (8) pilosinine. Based on the dissociation patterns of
the main compounds found in the extracts it was possible to separate the
identified alkaloids into three structurally related groups of compounds (Group
A = 1, 4 and 8; Group B = 2 and 3; Group C = 5, 6 and 7), which varied with
the seasons of the year. But, more interestingly, these results indicated that
these three groups could belong to intermediate, parallel or even competitive
pathways.
A preliminary metabolomic study, carried out with juvenile and adult
plants of jaborandi by 2D-1H nuclear magnetic resonance and electrospray
ionization mass spectrometry [ESI (+) – MS] and result analysis by principal
component analysis, showed that pilocarpine biosynthesis is not dependent on
the plant development stage while biosynthesis of pilosine occurs only in the
adult stage (Abreu and Mazzafera, unpublished results). Therefore, this is an
additional indication that different pathways are present in the synthesis of
imidazole alkaloids in jaborandi. More recently, other indirect support for this
conclusion came from a comparative study of the alkaloids of seven
Pilocarpus species where the presence of already known alkaloids was
confirmed and novel alkaloids were detected (Sawaya et al., 2010). In this
work a HPLC method coupled with ESI-MSn developed for qualitative and
quantitative analysis (Sawaya et al., 2008) was used, which allowed us to
observe a remarkable variation in the alkaloid profiles and contents in the
seven species. For example, although P. microphyllus is considered to have the
highest concentration of pilocarpine and is its commercial source, three other
species were found to contain higher concentrations of pilocarpine (P.
jaborandi, P. racemosus and P. trachyllophus). On the other hand P.
jaborandi did not contain pilosine or its isomers and P. spicatus had only
traces of pilocarpine. Therefore, these species may be used as tools to study
the biosynthetic pathway of these alkaloids, since species with different
alkaloid contents and composition may be crossed and the F1 plants analyzed
in their alkaloid constitution. Additionally, the recent method developed to
68 Alexandra C. H. F. Sawaya, Ilka N. Abreu, Nathalia L. Andreazza et al.

separate the different alkaloids of jaborandi (Sawaya et al., 2008) may now be
used in studies with radioactive labeled amino acids furnished to these species.
1.2. Biotechnological Applications

Cell suspension cultures using bioreactors are among the promising

techniques applied to the production of secondary metabolites of economical
importance. Its main advantage over other techniques is that it allows rapid
cell proliferation in short intervals of time and the control of the medium
culture, enabling changes of the medium composition or addition of
compounds that can stimulate the production of the target molecule. However,
to become economically feasible some aspects must be satisfied: 1) the
demand; 2) the price of the compound of interest; 3) the culture yield and 4)
the culture costs. A high demand may be masked by a low price or a low
demand would be compensated by a high price of the compound. The culture
costs are directly related with culture yield. In this aspect, compounds of
interest released in the medium certainly decrease the culture costs, as they can
be recovered by medium replacement. Also, some of the cells can be used as
starting culture avoiding the initial steps of culturing, when contamination can
be high (Verpoorte and Memelink, 2002; Verpoorte et al., 1993; Verpoorte et
al., 1999). Another important advantage of cell suspension cultures over plants
is that the content of the compound in the plant tissues may vary significantly
according the cultivating conditions and long periods of time may be
necessary until the plant reach the harvest stage. Additionally, unwanted
compounds (or contaminants) may be high in green tissues requesting
additional purification steps (Verpoorte et al., 1993; Verpoorte et al., 1999).
Several studies were published on the regulation of alkaloid biosynthesis
by different abiotic factors, such as light, temperature, gas composition,
salinity, osmotic stress, jasmonic acid, pH alterations and nutritional stresses
(Baricevic et al., 1999; Godoy-Hernandez et al., 2000; Li and Liu, 2003;
Stafford et al., 1986; Van der Fits et al., 2000). In this sense P. microphyllus
calli were used to study how different biotic factors could affect pilocarpine
production and release in the medium (Abreu et al., 2005). The calli
maintained in the dark released the greatest quantities of pilocarpine into the
medium. Methyljasmonate inhibited the release of pilocarpine into the
medium. Neutral pH (6.8), absence and excess of N, excess of P, and 0.75 mM
of histidine and threonine induced the highest production of the alkaloid.
Growth medium with a pH of 6.8 induced the highest release o pilocarpine in
the medium. Other pH values (4.8 and 5.8) reduced pilocarpine production by
 Pilocarpine and Related Alkaloids in Pilocarpus Vahl (Rutaceae) 69

20% as compared with pH 6.8 and less of the alkaloid was released into the
medium.
Cell suspension cultures established from the leaf petioles of different P.
microphyllus plants showed a marked difference among the obtained lineages
regarding alkaloid production and release into the medium (Abreu et al.,
2007a). This might be attributed to genetic variation as reported for
pilocarpine in jaborandi (Sandhu et al., 2005). Primary cultures of jaborandi
cells were shown to produce the highest amount of pilocarpine and also
pilosine, but decreased with sub-culturing (Abreu et al., 2007a). However,
after 24 sub-cultures, the production of alkaloids remained constant. This
study also showed that cell suspension culture extracts had the same alkaloid
profile as leaf extracts.
Another study used cell suspension cultures of jaborandi to verify the
effects of initial pH values on the production and release of pilocarpine and
other imidazole alkaloids in the medium (Andreazza et al., 2008). Two cell
line were grown in liquid culture with initial pH values of 4.8, 5.8, 6.8, 7.8, 8.8
and 9.8 and the alkaloid contents in the cells and medium was followed for 30
days. Pilocarpine reached the highest content per flask when the initial pH was
8.8 and 9.8 while for pilosine this was observed at pH 7.8. At all pH values
there was release of pilocarpine into the medium but always in lower amount
than in the cells.
The results obtained so far for the release of pilocarpine into the medium
is probably related to the way jaborandi cells control the transport of this
alkaloid through the membranes, as well as the cell compartment it is
accumulated in. Histochemical and cell fractionation tests indicated that
pilocarpine very probably accumulates in the vacuole of jaborandi cells
(Andreazza, 2009). This was reinforced by the fact that cells could grow in
suspension cultures with very high concentrations of exogenous pilocarpine
added to the medium, indicating a detoxifying mechanism of vacuole
accumulation, as the alkaloid internal cell concentration increased during
cultivation. Additionally, studies with cell suspension cultures with or without
addition of pilocarpine to the medium and using several ABC transporters
inhibitors showed the participation of these proteins controlling the transport
of pilocarpine in and out of the cells (Andreazza, 2009). Therefore, a strategy
to increase the release of pilocarpine into the medium would be the over-
expression of specific genes for specific ABC proteins, as suggested for other
systems (Oksman-Caldentey and Inze, 2004).
70 Alexandra C. H. F. Sawaya, Ilka N. Abreu, Nathalia L. Andreazza et al.

O
O O
O
O O
N
N N

N N
N
OH
A B C
O
O O
O O
O N
N
N

N N
N
OH OH OH
D E F
O
O O
O
O O
N
N N

N N N

HO G HO H I
O
O O O
O O
N
N N

N
N N
J H K H L
O
O O O
O
O O
N N
HN

N N O N

M N O
OH
O O
O
O
N

N N

N
N N
O
P Q R

Figure 2. Imidazole alkaloids found in Pilocarpus: A. pilocarpine, B. isopilocarpine, C.

pilosine, D. isopilosine, E. epi-isopilosine, F. epi-pilosine, G. piloturin, H. epi-
isopiloturin, I. pilosinine, J. dehydropilosinine, K. pilocarpidine, L. isopilocarpidine,
M. 13-nor-7(11) dehydropilocarpine, N. anhydropilosine, O. 4,6-dehydro-1,2,4,5-
tetrahydro-2,5- dioxopilocarpine, P. 3-(3-methyl-3H-imidazol-4-ylmethyl)-1-phenyl-
but-3-en-1-one, Q. 3-Hydroxymethyl-4-(3-methyl-3H-imidazol-4-yl)1-phenyl-butan-1-
one, R. 3-Benzoyl-4-(3-methyl-3H-imidazol-4-ylmethyl)-dihydro-furan-2-one.
 Pilocarpine and Related Alkaloids in Pilocarpus Vahl (Rutaceae) 71

2. ALKALOIDS FROM PILOCARPUS,

PLANT SOURCES AND KNOWN ACTIVITY
Most of the alkaloids isolated from the Pilocarpus genus are imidazole
alkaloids; several of which are epimers, leading to several studies reporting
their exact configuration. (Hill and Barcza, 1966; Link and Bernauer, 1972;
Link et al., 1974; Tedeschi et al., 1974). Methods to synthesize these alkaloids
have been suggested over the years, (Braun et al., 2004; Link and Bernauer,
1972; Link et al., 1974) but the overall yields were too low to be commercially
feasible. Different methods, proposed more recently for the synthesis of
pilocarpine, iso-pilocarpine and pilosinine report a yield of 37% of the racemic
alkaloid pilosinine (Davies et al., 2009a) and a 30% yield of pilocarpine nitrate
(Davies et al., 2009b). If these novel synthesis methods of pilocarpine prove to
be economically attractive still waits to be seen. For the moment, leaves of
species of Pilocarpus continue to be the source of pilocarpine. Other non-
imidazole alkaloids have been found in this genus.

2.1. Pilocarpine and Isopilocarpine

References to pilocarpine remote to the late 19th century, isolated from

jaborandi (Gerard, 1875; Hardy, 1875; Park, 1883). Pilocarpine (Fig. 2 A)
affects the central nervous system. It is a direct cholinergic; stimulating the
parasympathetic system and thus affecting the bladder, tear ducts, salivary and
sweat glands (Santos and Moreno, 2004). It has been used for the treatment of
glaucoma for many years (Goodman and Gilman, 2001) and is also applied to
the treatment of xerostomy, mainly in patients who have undergone
radiotherapy in the head and neck area (Valdez et al., 1993).
Since pilocarpine is a non-selective muscarinic agonist, it is not routinely
used orally in humans. But, due to its high affinity for muscarinic receptors, it
is used to develop an experimental model of temporal lobe epilepsy in
laboratory animals (Uva et al., 2008). Studies have shown that it does not
easily permeate the blood brain barrier and the exact mechanism of its pro-
epileptic effect has not been fully determined.
Its isomer, isopilocarpine (Fig. 2B), does not have any reported
pharmacological activity. In aqueous solution, pilocarpine is known to
hydrolyze to pilocarpic acid and to epimerize to isopilocarpine (Merbel et al.,
1998). As the standard extraction of alkaloids contains a step in which the
72 Alexandra C. H. F. Sawaya, Ilka N. Abreu, Nathalia L. Andreazza et al.

alkaloids are dissolved in acidic water, both isomers may exist naturally in the
plants, but may also be formed during the extraction process.
Pilocarpine contents from several species of Pilocarpus: P. microphyllus,
P. carajaensis, P. trachyllophus, P. pennatifolius, P. jaborandi, and P.
racemosus. (Sawaya et al., 2010), were determined, presenting concentrations
between 2 and 70% of the total alkaloids. Isopilocarpine was also present in
lower concentrations. Pilocarpine was not found in P. spicatus (Kaastra, 1982;
Sawaya et al., 2010) nor was its isomer, isopilocarpine (Sawaya et al., 2010).
Several alkaloids have been reported in P. grandiflorus, but not pilocarpine
(Souza et al., 2005).
Commercially, pilocarpine is mainly extracted from P. microphyllus
planted in the state of Maranhão, in the Brazilian tropics (Pinheiro, 2002).
Other species may be better adapted in different countries and climates, and
efforts have been made to determine their pilocarpine contents. In Cuba, for
example, 0.6 g of pilocarpine was extracted from 1 Kg of P. racemosus leaves
(Payo Hill et al., 1995). P. pennatifolius grown in Argentina, had 0.4 to 0.5%
of total alkaloids in relation to dry leaf weight, with about 0,25% of
pilocarpine (Lucio et al., 2002). Although several imidazole alkaloids have
antimicrobial activity (de Luca, 2006) this does not seem to be the case with
pilocarpine, as pilocarpine extracted from P. racemosus in Guadalupe
(Caribbean) was not active against mycobacterium (Rastogi et al., 1998).

2.2. Pilosine and Isomers

Another imidazole alkaloid found in Pilocarpus is pilosine (Fig. 2C)

which has three other theoretically possible isomers (Figs. 2 D, E and F),
however only isopilosine and epiisopilosine have been isolated (Fig. 2 D and
E) (Tedeschi et al., 1974). Furthermore, piloturin and epi-isopiloturin (Figs. 2
G and H) which are linked to the imidazole ring at the C4 (instead of at the C5
as in pilosine) have also been extracted from P. microphyllus leaves
(Voigtlander et al., 1978). Of these, only epiisopilosine has been screened for
pharmacological and toxicological activity. At high doses it is a stimulant of
the parasympathetic system, much like pilocarpine. However, it’s DL 50 is
twice that of pilocarpine, indicating that it is less toxic (Lucio et al., 2000). No
pharmacological studies of the other isomers were found.
Pilosine and some isomers were found in P. microphyllus leaves and one
of its isomers in P. carajaenis (Sawaya et al., 2010) but none of them in P.
jaborandi. This conflicts with a previous reports of these compounds in P.
 Pilocarpine and Related Alkaloids in Pilocarpus Vahl (Rutaceae) 73

jaborandi (Tedeschi et al., 1974). As several of the Pilocarpus species are

commonly known as jaborandi it is possible that the species was incorrectly
identified at that time. Pilosine was also produced, and released into the
medium by cell cultures of P. microphyllus (Abreu et al., 2007a; Andreazza et
al., 2008).

2.3. Pilosinine

Pilosinine (Fig. 2 I) is structurally similar to both pilocarpine and iso-

pilocarpine, and has been proposed as a precursor in the synthesis of these
alkaloids (Link and Bernauer, 1972). Its melting point and some other
characteristics were further determined (Voigtlander et al., 1978). A recent 6-
step method has been devised that results in a 37% yield of pilosinine (Davies
et al., 2009a). Pilosinine was found in leaves of P. microphyllus (Abreu et al.,
2007b). No biological activity has been attributed to this alkaloid.

2.4. Dehydropilosinine

Also structurally similar to pilosinine, dehydropilosinine (Fig. 2 J) was

characterized while seeking to synthesize other imidazole alkaloids (Link and
Bernauer, 1972). It was also detected in leaves of P. carajaensis , P. spicatus,
P. trachyllophus, P. pennatifolius, P. jaborandi and P. racemosus, but not of
P. microphyllus (Sawaya et al., 2010), indicating that it is quite common in
this genus. No biological activity has been reported.

2.5. Pilocarpidine and Isopilocarpidine

Pilocarpidine and its isomer, isopilocarpidine, were only found in P.

jaborandi leaves (Santos and Moreno, 2004; Sawaya et al., 2010). These
compounds (Figs. 2 K and L) were suggested as possible intermediaries in the
biosynthesis of pilocarpine and isomer (Cordell, 1981). No biological activity
of these compounds has been reported.
74 Alexandra C. H. F. Sawaya, Ilka N. Abreu, Nathalia L. Andreazza et al.

2.6. 13-nor-7(11) Dehydropilocarpine

This alkaloid (Fig. 2 M) was first isolated from P. trachyllophus roots,

together with pilocarpine (Andrade-Neto et al., 1996). It has also been found
in the roots, basal stem and flowers of P. microphyllus (Abreu et al., 2007b),
as well as in the leaves of P. trachyllophus, P. carajaensis, P spicatus, P.
racemosus and P. pennatifolius (Sawaya et al., 2010). Although it appears in
several species of Pilocarpus, no biological activity has been reported.

2.7. Anhydropilosine and Isomers

The structure of anhydropilosine (Fig. 2 N) has been known for some time
(Tedeschi et al., 1974; Voigtlander et al., 1978) and it has been found in
several organs of P. microphyllus (Abreu et al., 2007b) as well as cell cultures
of this species (Abreu et al., 2007a). It has also been observed in leaves of P.
carajaensis and P spicatus , while two novel isomers (whose full structure has
not been fully elucidated yet) were also observed in P. carajaensis (Sawaya et
al., 2010). No biological activity has been reported for anhydropilosine or its
isomers.

2.8. 4,6-dehydro-1,2,4,5-tetrahydro-2,5- dioxopilocarpine

This alkaloid was isolated from P. grandiflorous (Souza et al., 2005).

While other alkaloids and phenolic compounds from this plant exhibited
antifungal activity, 4,6-dehydro-1,2,4,5-tetrahydro-2,5- dioxopilocarpine (Fig.
2O) did not present this activity.

2.9. Novel Imidazole Alkaloids from Pilocarpus

Several novel imidazole alkaloids have been extracted from species of

Pilocarpus, whose full structure and biological activity have not been
determined. Direct insertion ESI-MS fingerprints of alkaloid extracts from
diverse organs of P. microphyllus (Abreu et al., 2007b) and of cell cultures of
this species (Abreu et al., 2007a) showed the same three ions of m/z 241, 259
and 285. High resolution mass studies confirmed their molecular formula that
led to the proposed structures (Figs. 2 P, Q and R) and these alkaloids were
 Pilocarpine and Related Alkaloids in Pilocarpus Vahl (Rutaceae) 75

designated by their chemical names: 3-(3-methyl-3H-imidazol-4-ylmethyl)-1-

phenyl-but-3-en-1-one; 3-Hydroxymethyl-4-(3-methyl-3H-imidazol-4-yl)1-
phenyl-butan-1-one and 3-Benzoyl-4-(3-methyl-3H-imidazol-4-ylmethyl)-
dihydro-furan-2-one, respectively.
A later study, with HPLC-MS of leaf extracts of P. microphyllus, showed
that the compound detected as [M+H]+ of m/z 259 [3-Hydroxymethyl-4-(3-
methyl-3H-imidazol-4-yl)1-phenyl-butan-1-one] was in fact a mixture of two
isomers (Sawaya et al., 2008). One or both of these isomers were also detected
in leaves P. carajaensis, P. trachyllophus and P. racemosus (Sawaya et al.,
2010). A single peak of m/z 285 was observed in HLPC-MS of P.
microphyllus leaves (Sawaya et al., 2008; Sawaya et al., 2010) indicating that
only one isoform of 3- Benzoyl-4-(3-methyl-3H-imidazol-4-ylmethyl)-
dihydro-furan-2-one was present.

2.10. Non-imidazole Alkaloids

Although the main alkaloids found in Pilocarpus are of the imidazole

moiety, a few non-imidazole alkaloids were isolated from specific species.
N,N-dimethyl-5-methoxy-triptamine and N,N-dimethyl-triptamine were
isolated from P. organensis and are active on the central nervous system
(Santos and Moreno, 2004). Platidesmine, (1H)-4-methoxy-2-quinolone and
dictamine were extracted from P. grandiflorus, and inhibited fungal growth
(Souza et al., 2005).

3. CONCLUDING REMARKS
Although several attempts to synthesize pilocarpine have been carried out
over the years the main source of this alkaloid continues to be the leaves of P.
microphyllus. Therefore knowledge of the routes leading to its biosynthesis
and ways to increase pilocarpine production are of utmost interest. There are,
however, many questions to be answered in this respect. Furthermore, the
possibility of pilocarpine biosynthesis in cell cultures should also be further
explored.
Several other alkaloids have been isolated and identified in the Pilocarpus
genus, but only few have been tested for pharmacological activity and none
are used commercially. Recent studies have indicated the presence of novel
76 Alexandra C. H. F. Sawaya, Ilka N. Abreu, Nathalia L. Andreazza et al.

imidazole alkaloids in P. microphyllus and other species of Pilocarpus whose

structure and biological activities have not yet been determined. The
Pilocarpus genus should prove to be a rich source of new, and possibly
bioactive, alkaloids in the years to come.

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