Diosgenin quantification by HPLC in a Dioscorea polygonoides tuber collection from colombian flora

J. Braz. Chem. Soc., Vol. 18, No. 5, 1073-1076, 2007. Printed in Brazil - ©2007 Sociedade Brasileira de Química 0103 - 5053 $6.00+0.00 Jaime Niño,* Diego A. Jiménez, Oscar M. Mosquera and Yaned M. Correa Grupo de Biotecnología - Productos Naturales, Escuela de Tecnología Química, Universidad Tecnológica de Pereira, La Julita, A.A. 97 Pereira, Colombia As concentrações de diosgenina em uma coleção de tubérculo de Dioscorea polygonoides do Eje Cafetero (EC, Colombia) foram determinadas por cromatografia líquida de alta eficiência. Os teores obtidos variaram de 0,02 a 2,64%. A maior concentração de diosgenina (2,64%) foi obtida de um tubérculo coletado perto de Salento (Quindio). A média de recuperação desta foi de 97%. A identificação foi feita por cromatografia gasosa-espectrometria de massas (CG-EM) e por coeluição com o padrão autentico. Os resultados destes estudos foram importantes uma vez que os teores de diosgenina obtidos se mostraram superiores quando comparados aqueles relatados para outras espécies medicinais de Dioscorea, tornando Dioscorea polygonoides uma fonte potencial de diosgenina. The diosgenin concentrations in a Dioscorea polygonoides tuber collection from the Eje Cafetero (EC, Colombia) were determined by HPLC and their percentages ranged from 0.02 to 2.64%. The highest diosgenin concentration (2.64%) was obtained in a tuber collected near Salento (Quindío). The average of diosgenin recovery was 97%. Diosgenin was identified by gas chromatography-mass spectrometry (GC-MS) and by coelution with authentic diosgenin standard in both HPLC and GC-MS techniques. The results of this study are important since comparatively they are higher than most of those reported on other medicinal Dioscorea species, making Dioscorea polygonoides a potential new source of diosgenin. Keywords: bioprospection, Colombian biodiversity, Dioscorea, diosgenin screening, steroidal sapogenin Introduction The most important families that biosynthesize steroidal saponins are Agavaceae (genus Agave), Dioscoreaceae (genus Dioscorea) and Liliaceae (genera Allium, Asparagus, Lilium). 1 Yams belong to genus Dioscorea (Dioscoreaceae family) with near 400 species growing in the tropical and subtropical wet areas around the world. This family is characterized by the production of subterranean or aerials tubers. Usually its species are climbing vines and many of them are dioicous.2 Several Dioscorea species are economically important as staple food,3,4 and others are used for the production of steroidal saponins, which on hydrolysis render sapogenins, such as diosgenin (Figure 1), which is important as starting material for the production of corticosteroids, sexual hormones, oral contraceptives as well as other steroids via hemisynthesis.5-7 *e-mail: Dioscorea polygonoides Humb. et. Bonpl. ex Willd. belongs to the Dioscoreaceae family, genus Dioscorea, subgenus Eudioscorea and to the section Lynchnostemon, which is well distributed from Mexico to Brazil, crossing by Colombia and the Antilles.2,8 From D. polygonoides two steroidal saponins, diospolysaponin A and prosapogenin A of dioscin have been isolated and characterized. The last compound showed cytotoxic activity against the HSC-2 (IC50 3.4 μg mL-1) cell line which was as potent as the positive control, doxorubicin (IC50 2.5 μg mL-1).9 In addition, three O O HO Figure 1. Diosgenin. Short Report Diosgenin Quantification by HPLC in a Dioscorea polygonoides Tuber Collection from Colombian Flora 1074 Diosgenin Quantification by HPLC in a Dioscorea polygonoides Tuber Collection J. Braz. Chem. Soc. new polyhydroxylated spirostanol saponins were isolated from D. polygonoides tubers and their structure determined on the basis of extensive spectroscopic analysis.10 To date, diosgenin and related sapogenins are commercially available from tubers of different Dioscorea species; however, recent experimental evidence has showed that diosgenin has proved human health benefit.11 These discoveries have increased diosgenin demand world wide and motivated the search for new and renewable sources for this important raw material for the pharmaceutical industry. The aim of this study was to quantify by high pressure liquid chromatography (HPLC) the levels of diosgenin in a collection of seventy four tubers of D. polygonoides from different localities of the Eje Cafetero (EC, Colombia). ground to a fine powder, as described by Mahato et al.12 Next, for each colleted tuber 10.00 g aliquot were taken and defatted with n-hexane in a Soxhlet extractor. The degreased plant materials were extracted three times with methanol in Soxhlet, and the combined methanol extracts were concentrated to dryness at reduced pressure. After that, each methanol extract was resuspended in 10 mL of water HPLC grade and partitioned three times with portions of 6 mL of n-butanol saturated with water, to obtain the crude saponins.13 Finally, 50 mg of each butanol extract were hydrolyzed by treatment with the enzyme naringinase to obtain the crude sapogenins, following the procedure described by Niño et al.9 Experimental Diosgenin quantification was performed on a HPLC instrument applying the external standard method, through a diosgenin calibration curve with six points in a concentration range between 50-300 mg L -1 . The experimental conditions were an isocratic binary system of acetonitrile/water (90:10), a flow rate of 1 mL min-1 and a temperature of 35 °C. Detection was performed at 194 nm, according to the procedure described by Oncina et al.14 The diosgenin concentrations in the different samples were calculated through a regression analysis from the peak area and the known concentrations of authentic diosgenin samples and are the average of three consecutive readings for each tuber sample. General experimental procedures The solvents n-hexane, chloroform, methanol, ethanol, n-butanol and acetonitrile used for the steroidal sapogenin extraction and analysis were purchased from Mallinckrodt (Phillipsburg, NJ, USA); diosgenin [CAS: 512-04-9] standard and the enzyme naringinase (E.C.: 232-962-4) were purchased from Sigma (St. Louis, MO, USA). For diosgenin quantification a HPLC Hewlett Packard instrument model HP-1100 (Palo Alto, CA, USA) equipped with the software version ChemStation A.06.01, a diode array detector (DAD-UV) was used; in addition, a Hypersil ODS C18 column (250 × 4.0 mm, 5 μm) and a 20 μL Rheodyne manual injector were used. Diosgenin quantification Results and Discussion Tuber collection Plant material Tubers of D. polygonoides were collected during the period between November 2002 and November 2003 at different zones in the EC. This area is constituted by the departments of Caldas, Quindío and Risaralda (Colombia), and they are located on the Central Andean mountain chain, with an extension area of 13.893 km 2, with different altitudinal zones (900–5400 m) and high annual precipitation. Plant samples were authenticated by the authors, see Table (...truncated)