Enzymatic Extraction of Pilocarpine from Pilocarpus jaborandi

Many of the alkaloids used as drugs today are from natural sources. These drugs have been modified to produce analogs for clinical use. One of these alkaloids, pilocarpine, is an imidazole alkaloid found in plants of the genus Pilocarpus (Fig. 1). Plants of this genus are designated by the name jaborandi but only Pilocarpus microphyllus, which accumulates the highest pilocarpine content, is considered as true jaborandi [17, 22]. Jaborandi growing as a shrub is found in the understory of the pre Amazonian rain forest and occurs more intensively in the state of Maranhão [18, 22]. Pilocarpine has important pharmaceutical properties. It is used to reduce the intraocular pressure in the treatment of glaucoma [14], as a stimulant of salivation and perspiration, and recently has been prescribed for the treatment of xerostomia, which a symptom refers abnormal dryness of the mouth due to the reduction of saliva production [5]. In spite of the importance of the plant and the pharmacological activity of pilocarpine, only a few reports have been published for this alkaloid in Pilocarpus [2, 3]. Jaborandi leaves are the only known source of pilocarpine, an imidazole alkaloid probably derived from histidine [6]. Several solvent-based extraction protocols for pilocarpine have been reported. Unfortunately, these methods often suffers from low extraction yields, long extraction times and potential existence of trace organic solvent in final products which decrease the product quality [25]. Several supercritical fluid and organic solvent-based extraction protocols to extract pilocarpine and other alkaloids have been reported [3, 19]. However, the use of organic solvents for Pilocarpine is an imidazole alkaloid, found exclusively in the Pilocarpus genus, with huge pharmaceutical importance. In order to extract pilocarpine from Pilocarpus jaborandi, environmentally friendly enzyme-assisted extraction was applied. Viscozyme L, a commercially available enzyme cocktail, was used for the study. The conditions for extraction were optimized on the basis of substrates, enzymes, temperatures and pHs. Optimum conditions for extraction with the highest yield were 30 h reaction of 100 mg substance at 45oC in 40 ml of 50 mM acetic acid, pH 4. A 10% enzyme concentration was found to be the best for extraction. Total pilocarpine content after extraction was analyzed by HPLC. The total pilocarpine content (1.14 μg/mg) obtained from Viscozyme L treatment was 3.08-fold greater than those of the control treatment (0.37 μg/mg).