Pilocarpine and Related Alkaloids in Pilocarpus Vahl (Rutaceae)

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,* 1Plant Biology Department, Institute of Biology, State University of Campinas, UNICAMP, CP 6109, Campinas, 13083-970, SP, Brazil 2Scottish Crop Research Institute, Department of Plant Products and Food Quality, Invergowrie, Dundee, DD2 5DA, Scotland, UK 3Thomson 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: [email protected] 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 CH R N 3 OH + N OR NH O N 2 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 N O O 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. epiisopiloturin, 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 isopilocarpine, 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.

4. REFERENCES

Abreu, I.N., Andreazza, N.L., Sawaya, A.C.H.F., Eberlin, M.N. and Mazzafera, P. (2007a) Cell suspension as a tool to study the biosynthesis of pilocarpine in Jaborandi. Plant Biology. 9, 793-799. Abreu, I.N., Mazzafera, P., Eberlin, M.N., Zullo, M.A.T. and Sawaya, A.C.H.F. (2007b) Characterization of the variation of the imidazole alkaloid profile of Pilocarpus microphyllus in different seasons and parts of the plant by electrospray ionization mass spectrometry fingerprinting and identification of novel alkaloids by tandem mass spectrometry. Rapid Commun. Mass Spectr. 21, 1205-1213. Abreu, I.N., Sawaya, A.C.H.F., Eberlin, M.N. and Mazzafera, P. (2005) Production of pilocarpine in callus of Jaborandi (Pilocarpus microphyllus Stapf). In Vitro Cellular Developmental Biology – PLANT. 41, 806-811. Andrade, P., Willoughby, R., Pomponi, S.A. and Kerr, R.G. (1999) Biosynthetic studies of the alkaloid, stevensine, in a cell culture of the marine sponge Teichaxinella morchella. Tetrahedron Letters. 40, 4775- 4778. Andrade-Neto, M., Mendes, P.H. and Silveira, E.R. (1996) An imidazole alkaloid and other constituents from Pilocarpus trachyllophus. Phytochemistry. 42, 885-887. Andreazza, N.L. (2009) Transporte de pilocarpina em suspensões celulares de Pilocarpus microphyllus. Universidade estadual de Campinas. Andreazza, N.L., Abreu, I.N., Sawaya, A.C.H.F., Eberlin, M.N. and Mazzafera, P. (2008) Production of imidazole alkaloids in cell cultures of jaborandi as affected by the medium pH. Biotechnology Letters. 31, 607- 614. Avancini, G., Abreu, I.N., Saldaña, M.D.A., Mohamed, R.S. and Mazzafera, P. (2003) Induction of pilocarpine formation in jaborandi leaves by salicylic acid and methyljasmonate. Phytochemistry. 63, 171-175. Baricevic, D., Umek, A., Kreft, S., Maticic, B. and Zupancic, A. (1999) Effect of water stress and nitrogen fertilization on the content of hyoscyamine Pilocarpine and Related Alkaloids in Pilocarpus Vahl (Rutaceae) 77

and scopolamine in the roots of deadly nightshade (Atropa belladonna). Environmental and Experimental Botany. 42, 17-24. Braun, M., Buhne, C., Cougali, D., Schaper, K. and Frank, W. (2004) Convergent diasteroselective synthesis of isopilocarpine by one-pot Michael-addition-alkylation reaction. Synthesis. 17, 2905-2909. Cordell, G.A. (1981) Introduction to alkaloids: a biogenetic approach. New York, John Wiley & Sons. Davies, S.G., Roberts, P.M., Stephenson, P.T. and Thomson, J.E. (2009a) Syntheses of the racemic jaborandi alkaloids pilocarpine, isopilocarpine and pilosinine. Tetrahedron Letters. vol. 50, 3127,3509-3512. Davies, S.G., Robets, P.M., Stephenson, P.T., Storr, H.R. and Thomson, J.E. (2009b) A practical and scalable total synthesis of the jaborandi alkaloid (+)-pilocarpine. Tetrahedon. 65, 8283-8296. de Luca, L. (2006) Naturally occurring and synthetic imidazoles. Their chemistry and their biological activities. Current Medical Chemistry. 13, 1-23. Dewick, P.M. (1997) Medicinal natural products: a biosynthetic approach. New York, John Wiley & Sons. Gerard, A.W. (1875) Alkaloid and active principles of jaborandi. Pharmaceutical Journal. 5, 865. Godoy-Hernandez, G.C., Vazquez-Flota, F.A. and Loyola-Vargas, V.M. (2000) The exposure to trans-cinnamic acid of osmotically stressed Catharanthus roseus cells cultured in a 14-L bioreactor increases alkaloid accumulation. Biotechnology Letters. 22, 921-925. Goodman, L.S. and Gilman, A. (2001) The pharmacological basis of therapeutics, 10th edition. New York, McGraw Hill Companies Inc. Hardy, M.E. (1875) Sur le jaborandi (Pilocarpus pinnatus). Bulletin Societe Chimi Paris. 24, 497-500. Hemscheidt, T. and Spenser, I.D. (1991) Biosynthesisof anosmine, an imidazólico alkaloid of the orchid Dendrobium parishii. Journal of Chemical Society - Chemical Communications. 7, 494-497. Hill, R.K. and Barcza, S. (1966) Sterochemistry of the Jaborandi alkaloids. Tetrahedon. 22, 2889-2893. Holmstead, B., Wassen, S.H. and Schultes, R.E. (1979) Jaborandi: an interdisciplinary appraisal. Journal of Ethnopharmacology. 1, 3-21. IBAMA (1992) - Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis. Portaria n° 06N, Diário Oficial, Brasília, p. 870- 872, janeiro 15, 1992. 78 Alexandra C. H. F. Sawaya, Ilka N. Abreu, Nathalia L. Andreazza et al.

Joseph, C.J. (1967) Revisão sistemática do gênero Pilocarpus (ssp. brasileiras). Mecânica Popular. October, 1-9. Kaastra, R.C. (1982) Pilocarpinae. Flora Neotropica. Monograph number 33. New York Botanical Garden, New York. Li, Z.H. and Liu, Z.J. (2003) Effect of NaCl on growth, morphology, and camptothecin accumulation in Camptotheca acuminata seedlings. Canadian Journal of Plant Science. 83, 931-938. Link, H. and Bernauer, K. (1972) Über die Synthese der Pilocarpus-alkloide isopilosin und pilocarpin, sowie die absolute konfiguration dês (+)- Isopilosins. Helvetica Chimica Acta. 55, 1053-1062. Link, H., Bernauer, K. and Oberhans, W.E. (1974) Link H, Bernauer K, Oberhans WE 1974. Configuration of Pilocarpus alkaloids. Helv Chim Acta 57: 2199-2200. Helvetica Chimica Acta. 57, 2199-2200. Lucio, E.M.R., Rosalen, P.L., Sharapin, N. and Souza-Brito, A.R.M. (2000) Avaliação toxicológica e screening hipocrático de epiisopilosine, alcalóide secundário de Pilocarpus microphyllus Staph. Revista Brasileira de Farmacognosia. 9, 23-35. Lucio, E.M.R., Sharapin, N. and França, H.S. (2002) Estudo de alcalóides de Pilocarpus pennatifolius Lemaire. Revista Brasileira de Farmacognosia. 12, 130-131. Merbel, N.C., Tinke, A.P., Oosterhuris, B., Jonkman, J.H.G. and Bohle, J.F. (1998) Determination of pilocarpine, isopilocarpine, pilocarpic acid and isopilocarpic aid in human plasma and urine by high performance liquid chromatography and tandem spectrometric detection. Journal of Chromatography B. 708, 103-112. Oksman-Caldentey, K.M. and Inze, D. (2004) Plant cell factories in the post- genomic era: New ways to produce designer secondary metabolites. Trends in Plant Science. 9, 433-440. Oliveira, P.D. (2007) Filogenética de Pilocarpinae (Rutaceae). PhD Thesis, Universidade de São Paulo. Park, R. (1883) On the use of Jaborandi or pilocarpine in the collapse of scarlatina maligna. Lancet. 121, 1041. Payo Hill, A., Dominicis, M.E., Oquendo, M. and Sarduy, R. (1995) Obtención de pilocarpina a partir de Pilocarpus racemosus Vahl. Revista Cubana de Farmacia. 29, 123-125. Pinheiro, C.U.B. (1997) Jaborandi (Pilocarpus sp., Tutaceae): a wild species and its rapid transformation into a crop. Econ. Bot. 51, 49-58. Pilocarpine and Related Alkaloids in Pilocarpus Vahl (Rutaceae) 79

Pinheiro, C.U.B. (2002) Extrativismo, cultivo e privatização do jaborandi (Pilocarpus microphyllus Stapf ex Holm.; Rutaceae) no Maranhão. Acta bot. bras. 16, 141-150. Rastogi, N., Abaul, J., Goh, K.S., Devallois, A., Philogene, E. and Bourgeois, P. (1998) Antimycobacterial activity of chemically defined natural substances form the Caribbean flora in Guadeloupe. FEMS Immunology and Medical Microbiology. 20, 267-273. Sandhu, S.S., Abreu, I.N., Colombo, C.A. and Mazzafera, P. (2005) Pilocarpine content and molecular diversity in Jaborandi. Scientia Agricola. 63, 478-482. Santos, A.P. and Moreno, P.R.H. (2004) Pilocarpus spp.: A survey of its chemical constituents and Biological activities. Brazilian Journal of Pharmaceutical Sciences. 40, 115-137. Sawaya, A.C.H.F., Abreu, I.N., Andreazza, N.L., Eberlin, M.N. and Mazzafera, P. (2008) HPLC-ESI-MS/MS of imidazole alkaloids in Pilocarpus microphyllus. Molecules. 13, 1518-1529. Sawaya, A.C.H.F., Gontijo Vaz, B., Eberlin, M.N. and Mazzafera, P. (2010) Comparative study of imidazole alkaloids in seven species of Pilocarpus. Submmited. Skorupa, L.A. (1996) Revisão Taxonômica de Pilocarpus Vahl (Rutaceae). PhD Thesis, Universidade de São PAulo. Sousa, M.P., Matos, M.E.O., Matos, F.J.A., Machado, M.I.L. and Craveiro, A.A. (1991) Constituintes químicos ativos de plantas medicinais brasileiras. Fortaleza, Brazil, Editora da Universidade Federal do Ceará. Souza, R.C., Fernandes, J.B., Vieira, P.C., Silva, M.F.G.F., Godoy, M.F.P., Pagnocca, F.C., Bueno, O.C., Hebling, M.J.A. and Pirani, J.R. (2005) New imidazole alkaloid and other constituents from Pilocarpus grandiflorus and their antifungal activity. Zeitschrift für Naturforschung B. 60, 787-791. Stafford, A., Morris, P. and Fowler, M.W. (1986) Plant cell biotechnology: a perpective. Enzyme Microbial Technology. 8, 578-587. SUDEMA (1970) - Superintendência do Desenvolvimento do Maranhão. Novo Zoneamento do Estado do Maranhão. São Luis, MA. Tedeschi, E., Kamionsky, J., Zeider, D. and Fackler, S. (1974) Isolation, characterization, and synthesis of trans-pilosine stereoisomers occurring in nature. Circular dicrooism and mass spectral studies. Journal of Organic Chemistry. 39, 1864-1879. Uva, L., Librizzi, L., Marchi, N., Noe, F., Bongiovammi, R., Vezzani, A., Janigro, D. and de Curtis, M. (2008) Acute induction of epilelptiform 80 Alexandra C. H. F. Sawaya, Ilka N. Abreu, Nathalia L. Andreazza et al.

discharges by pilocarpine in the in vitro isolated guinea-pig brain requires enhancement of blood-brain barrier permeability. Neuroscience. 151, 303- 312. Valdez, I.H., Wolff, A., Atkinson, J.C., Macynsky, A.A. and Foz, P.C. (1993) Use of pilocarpine during head and neck radiation therapy to reduce xerostomia and salivary dysfunction. Cancer. 71, 1848-1851. Van der Fits, L., Zhang, H., Menke, F.L.H., Deneka, M. and Memelink, J. (2000) A Catharanthus roseus BPF-1 homologue interacts with an elicitor-responsive region of the secondary metabolite biosynthetic gene Str and is induced by elicitor via a JA-independent signal transduction pathway. Plant Molecular Biology. 44, 675-685. Verpoorte, R. and Memelink, J. (2002) Engeneering secondary metabolite in plant. Current Opinion in Biotechnology. 13, 181-187. Verpoorte, R., R., v.d.H., Schripsema, J., Hoge, J.H.C. and Ten Hoopen, H.J.G. (1993) Plant cell biotechnology for the production of alkaloids: Present status and prospects. Journal of Natural Products. 56, 186-207. Verpoorte, R., vanderHeijden, R., tenHoopen, H.J.G. and Memelink, J. (1999) Metabolic engineering of plant secondary metabolite pathways for the production of fine chemicals. Biotechnology Letters. 21, 467-479. Vieira, R.F. (1999) Conservation of medicinal and aromatic plants in Brazil. pp 152-159 in Janick, J. (Ed) Perspectives on new crops and new uses. Alexandria, ASHS Press. Voigtlander, H.W., Balsam, G. and Engelhardt, M. (1978) Epiisopiloturin, ein Neues Pilocapus-Alkaloid. Archiv der Pharmazie. 311, 927-935.