Local production of 5-HTP from the seeds of griffonia simplicifolia

Griffonia simplicifolia is a stout, woody, climbing shrub growing to about 3m with greenish flowers and inflated black pods. The seeds contain substantial quantities of the amino acid, 5-hydroxytryptophan (5-HTP). Today, the seed of this climbing shrub is used in the treatment of fibromyalgia, headaches, insomnia, depression and stomach problems. Griffonia seeds continue to be exported from Ghana. Therefore it was considered prudent to develop a simple method of production of 5-HTP from the seeds of Griffonia in reasonable purity prior to export. This will add value to the export. This project, is to develop enhanced extraction and purification methods in the production of 5-HTP from the seeds of Griffonia simplicifolia. In addition, the kinetics of the degradation of the 5-HTP obtained was studied in order to predict the most the stable conditions of storage prior to export. Griffonia seeds that had been harvested and stored for 10 months were used for the project. The solvent system used for all extractions was water- methanol (50-50). This has been determined to be the optimum solvent for the extraction (Lemaire and Adosraku 2002). The amount of 5-HTP present in the seeds was determined by High Pressure Liquid Chromatography. Using the HPLC method for the direct assay of 5-HTP from the seeds of Griffonia developed by Lemaire and Adosraku, the content of the seeds was found to be 6.37 %w/w. However, a modified method which employs the use of solvent at 80 °C gave 5-HTP content of 8.98 %w/w. The effect of varying conditions on the extraction of 5-HTP from the seeds was investigated. The conditions investigated were temperature of extracting solvent, volume of extracting solvent and the particle size of the powdered seeds. In all instances, the amount of 5-HTP extracted was determined by HPLC using pure 5-HTP as reference. Generally it was observed that an increase in the temperature and volume of solvent provided an increase in yield of 5-HTP extracted. A reduction in particle size of the powdered seeds by crushing the seeds, also increases the amount of 5-HTP extracted. A combination of the effects of the above factors was found to yield the highest amount of 5-HTP. The optimum solvent composition for the recrystallisation of the extracted 5-HTP crystals was also investigated. The limitation here is that the solvents used must non- toxic and readily available in Ghana. Thus, various compositions of water and ethanol were used to recrystallise the 5-HTP. The composition found to be useful was water- ethanol (20-80). Using this solvent mixture, 5-HTP was obtained as approximately 92% pure. The percentage yield of 5-HTP(92% pure) was 6.14 %w/w of seed taken. The purified 5-HTP crystals were subjected to stability studies, assuming first order kinetics. The rate constant for the degradation was 1.059 E-03 day-1 at refrigerator temperature (approx. 5°C) as compared to 1.195 E-03 day-1 for that kept at room temperature. The t90 values for refrigerator temperature and room temperature were 99.62 days and 88.28 days respectively. The difference in shelf life was thus 11 days. This is considered economically not significant considering the cost of refrigeration. The ether insoluble constituents of the aqueous- methanolic seed extract were separated using preparative thin layer chromatography and studied. Two constituents apart from 5- HTP were isolated. One of the constituents is postulated to be amino methoxy indole-3- acetic acid from the spectroscopic data obtained. The other constituent could not be identified with certainty and further work will have to be done.

Source: KNUST