Ind. Eng. Chem. Res. 2004, 43, 5339-5346
5339
Recovery of Diosgenin from Dioscorea Rhizomes Using Aqueous Hydrotropic Solutions of Sodium Cumene Sulfonate Sanjay P. Mishra and Vilas G. Gaikar* Institute of Chemical Technology, University of Mumbai, Matunga, Mumbai 400019, India
Aqueous solutions of aromatic hydrotropes were investigated for cell permeabilization and extraction of dioscin from dioscorea rhizomes. The extracted dioscin was further hydrolyzed in the same hydrotropic solutions to diosgenin without significant decomposition to 3,5-diene, unlike in the conventional process. The parameters affecting the extraction of dioscin, such as the nature and concentration of the hydrotrope, the temperature, and the particle size, were optimized. Sodium cumene sulfonate was the most efficient hydrotrope for the extraction of dioscin and also for its hydrolysis to diosgenin at 353 K. Diosgenin precipitates from aqueous NaCS solutions with >95% purity at 293 K because of its poor solubility in aqueous solutions. Introduction Diosgenin (Figure 1) is a steroidal saponin belonging to the triterpene group that is a starting material for the synthesis of pharmaceutical products.1-4 It generally occurs in a combined form as a glycoside, called dioscin, throughout the tubers of the dioscorea plant but mostly in the stellar region of the tuber.1,5,6 Dioscin is a watersoluble compound, but diosgenin is almost insoluble in aqueous solutions. Dioscin is conventionally extracted from pulverized dioscorea rhizomes with alcohol and then subjected to acid hydrolysis under refluxing conditions for up to 12 h to produce diosgenin.7 An extraction process using supercritical carbon dioxide8 has been reported, and ultrasound treatment of the hydrolyzed dioscorea roots reportedly gave increased yields due to enhanced rates of mass transfer.9 As a part of our ongoing efforts in the development of aqueous-solution-based extraction process technology for natural products, we explore here the extraction of dioscin/diosgenin using “hydrotropy”. The hydrotropic phenomenon, which refers to the increased solubility of organic compounds in aqueous solutions of hydrotropes has recently been used successfully for the extraction of curcuminoids from Turmeric,10 piperine from Piper nigrum,11 and boswellic acids from Boswellia serrata resins.12 Hydrotropes, themselves, are highly watersoluble low-molecular-weight organic salts and show a strong concentration dependence in their ability to dissolve other organic compounds in aqueous solutions. Materials and Methods All chemicals, including p-toluenesulfonic acid (PTSA), sulfuric acid, hydrochloric acid, organic solvents, silica gel G, and iodine crystals (all AR grade), were procured from s. d. Fine Chemical Ltd., Mumbai, India. Pure diosgenin standard was provided by M/s. Belchem Industry Ltd., Mumbai, India, and dioscorea rhizomes were obtained from M/s. Herbotech Pharmaceutical Ltd., Amritsar, India. The hydrotropes, namely, sodium cumene sulfonate (NaCS), sodium xylene sulfonate (NaXS), and sodium * To whom correspondence should be addressed. Fax: 91-0 22-24145614. E-mail
[email protected].
Figure 1. Structures of diosgenin and dioscin and schematic representation of diosgenin decomposition to diene.
p-toluenesulfonate (NaPTSA), were obtained from Navdeep Chemicals Ltd., Mumbai, India, and were recrystallized from methanol and dried in an oven before use. Isobutyl benzene and n-butyl benzene obtained from Herdillia Chemicals Ltd., Mumbai, India, were sulfonated with concentrated sulfuric acid (98%) and then neutralized with aqueous NaOH to obtain sodium n-butyl benzene sulfonate (NaNBBS) and sodium isobutyl benzene sulfonate (NaIBBS). Solubility Studies. The solubility experiments were carried out in a fully baffled cylindrical glass vessel (100 cm3) equipped with a six-bladed turbine impeller (2 cm i.d.). This entire assembly was kept in a constanttemperature water bath with an accuracy of (0.1 °C.
10.1021/ie034091v CCC: $27.50 © 2004 American Chemical Society Published on Web 06/26/2004
5340 Ind. Eng. Chem. Res., Vol. 43, No. 17, 2004
Pure diosgenin was equilibrated with aqueous hydrotrope solutions in the concentration range 0.05-2.0 kmol/m3 for 4 h with vigorous stirring. Samples were withdrawn at regular intervals of time, and after centrifugation, the clear solution was diluted by water to precipitate diosgenin. The suspension was again centrifuged, and the solid residue was dissolved in chloroform for quantitative estimation spectrophotometrically as per the reported procedures.13,14 Extraction of Dioscin. Pulverized rhizomes of different mesh size were suspended in aqueous hydrotrope solutions, and the suspension was agitated vigorously at 1500 rpm. The rate of extraction became independent of the speed of agitation beyond 1500 rpm for 6- and 85-mesh size particles. Samples (0.5 cm3) were withdrawn at regular time intervals, and 0.05 cm3 of concentrated HCl was added to the sample. It was then kept in a water bath maintained at 353 K for the hydrolysis of dioscin to diosgenin for 40 min. The sample was finally cooled to 293 K and diluted to precipitate the diosgenin. The suspension was then centrifuged, and the solid residue was redissolved in 1 cm3 of chloroform for analysis. The final solution containing dioscin was treated in the same manner for the hydrolysis and subsequent recovery of diosgenin by dilution with water. The precipitated diosgenin was dried, weighed, and analyzed for purity.13,14 The decomposition of diosgenin to diene was separately studied at different temperatures with pure samples in 2.0 mol/dm3 aqueous NaCS solutions containing 1.0 mol/dm3 HCl. It was necessary to estimate the amount of diosgenin converted to diene, if any, during the hydrolysis. The total dioscin content in the raw material was separately determined by hydrolyzing pulverized rhizomes (10 g) in 2.0 mol/dm3 HCl solution for 3 h under total reflux conditions. The solid residue was filtered, washed with 10% (w/v) aqueous Na2CO3 solutions followed by water until the solution became neutral, and then dried in an oven at 70-80 °C. The dried and hydrolyzed rhizome was extracted using a Soxhlet extractor with petroleum ether (bp 60-80 °C) for 48 h. Diosgenin is soluble in the solvent under hot conditions and precipitates on cooling. The solid product was separated from the solution and analyzed spectrophotometrically and by thin-layer chromatography on silica G plates.13,14 The total recoverable diosgenin content of the raw material was 4.5% (w/w), and the dioscin content of the rhizomes was 9.4% (w/w). Results and Discussion The aromatic sulfonic acids and their sodium salts were selected as the hydrotropes for the solubility and extraction studies on the basis of the earlier work from this laboratory on solubilization and extraction of natural products.10-12 Hydrotropic solubilization is considered to be a collective molecular phenomenon, possibly occurring by intercalation or co-aggregation of the solute with the hydrotrope, and the self-aggregation of hydrotrope in aqueous solutions is considered prerequisite for the solubilization of other organic compounds.15,16 The solubilization capacity of a hydrotrope depends on the nature of the hydrophobic part of the hydrotrope, i.e., on the chain length as well as chain branching. The diosgenin solubility in water varies in the range of 4.4-9.7 mmol/m3 with an increase in temperature
Table 1. Solubility of Diosgenin (mol/m3) in Different Hydrotropes hydrotrope content (kmol‚m-3)
temperature (K) 293
303
313
323
333
343
Water 0.004 0.005 0.005 0.006 0.007 0.007
353
363
0.008
0.009
0.05 0.10 0.50 0.75 1.00 1.50 2.00
0.015 0.088 0.804 1.055 1.785 2.806 4.589
0.047 0.127 0.822 1.405 1.850 3.138 4.941
NaNBBS 0.036 0.023 0.117 0.109 0.911 1.301 1.666 1.976 1.959 2.802 3.220 4.302 5.395 7.715
0.05 0.10 0.50 0.75 1.00 1.50 2.00
0.011 0.062 0.424 0.655 0.852 1.750 2.029
0.025 0.066 0.445 0.699 0.965 1.791 2.248
NaIBBS 0.021 0.016 0.054 0.046 0.501 0.531 0.737 0.756 1.059 1.131 1.880 1.920 2.369 2.651
0.012 0.040 0.660 0.868 1.190 2.078 3.464
0.009 0.035 0.804 0.929 1.493 2.396 4.164
0.005 0.030 0.905 1.369 1.756 2.817 5.306
0.005 0.021 1.139 1.635 2.099 3.578 6.298
0.05 0.10 0.50 0.75 1.00 1.50 2.00
0.009 0.012 0.158 0.264 0.330 0.525 0.957
0.015 0.049 0.185 0.323 0.395 0.685 1.100
NaCS 0.012 0.082 0.043 0.035 0.301 0.386 0.453 0.597 0.596 0.685 0.960 1.105 1.613 2.081
0.006 0.029 0.450 0.696 1.099 1.578 2.674
0.005 0.029 0.484 0.894 1.203 1.952 3.164
0.005 0.023 0.582 1.028 1.349 2.066 3.487
0.004 0.020 0.728 1.286 1.495 2.425 4.296
0.10 0.50 0.75 1.00 1.50 2.00
0.005 0.013 0.019 0.022 0.034 0.056
0.006 0.015 0.021 0.026 0.036 0.067
NaPTSA 0.006 0.007 0.019 0.020 0.023 0.027 0.029 0.034 0.043 0.054 0.077 0.078
0.008 0.026 0.033 0.038 0.068 0.109
0.008 0.029 0.036 0.044 0.076 0.121
0.009 0.033 0.043 0.049 0.086 0.131
0.010 0.037 0.048 0.055 0.097 0.156
0.10 0.50 0.75 1.00 1.50 2.00
0.005 0.014 0.018 0.024 0.032 0.057
0.006 0.016 0.021 0.026 0.036 0.065
PTSA 0.007 0.007 0.017 0.020 0.024 0.027 0.030 0.034 0.047 0.051 0.078 0.084
0.008 0.022 0.030 0.036 0.065 0.094
0.008 0.026 0.035 0.043 0.073 0.117
0.009 0.032 0.040 0.046 0.083 0.134
0.010 0.036 0.048 0.051 0.096 0.150
0.018 0.098 1.452 2.280 2.790 4.770 8.334
0.010 0.008 0.006 0.089 0.054 0.029 1.586 1.590 1.856 2.464 2.886 3.212 2.969 3.900 4.399 5.794 6.292 6.623 9.064 10.590 12.249
from 293 to 363 K in an almost linear manner (Table 1). However, in the same temperature range, in aqueous solutions of different hydrotropes, diosgenin shows different solubilization behaviors below and above the characteristic minimum hydrotrope concentrations (MHCs) of the respective hydrotropes (Table 1).17,18 The solubility of diosgenin increased with increasing hydrotrope concentration and with temperature with all hydrotropes above their respective MHCs. Below the MHCs, however, the diosgenin solubility decreased with temperature, which is a significantly unusual behavior when compared to the reported solubilities of other compounds in aqueous hydrotrope solutions.10-12 Among the hydrotropes, NaCS showed a weaker solubilization effect than NaNBBS and NaIBBS but a much better ability than the other two hydrotropes, NaPTSA and p-toluenesulfonic acid (PTSA), to dissolve diosgenin. The alkyl chain attached to the aromatic ring, as the results indicate, plays a significant role in the solubilization process. NaNBBS showed the best solubilization of diosgenin. The solubility of diosgenin increased almost by 3 orders of magnitude in 2.0 kmol/m3 aqueous NaNBBS solutions. At concentrations below its MHC, however, the hydrotrope, being a strong electrolyte, salted-out diosgenin and the salting-out was more at increased temperatures. This salting-out also rules out any specific molecular complexation between diosgenin and the hydrotrope, although such complexation has
Ind. Eng. Chem. Res., Vol. 43, No. 17, 2004 5341
been indicated with some solutes.10 An association model19,12 for hydrotropic solubilization considers stepwise aggregation of hydrotrope molecules among themselves first and subsequent co-aggregation of a solute with these hydrotrope aggregates. The self-aggregation of a hydrotrope is favored by hydrophobic effect as governed by the hydrotrope’s structure and is opposed by the electrostatic repulsion between the charged headgroups. The aggregation model equations can be written in terms of the mass action law, provided that the aggregation constants of different steps are related someway with each other Kn
Hn-1 + H1 S Hn
Table 2. Hydrotrope-Hydrotrope (K2) and Hydrotrope-Solute (Ks) Association Parameters temperature (K)
Ks (m3/kmol)
K2 (m3/kmol)
NaNBBS
293 303 313 323 333 343 353 363
335.8 355.8 340.8 405.8 408.2 415.2 416.2 408.2
0.069 0.069 0.069 0.069 0.069 0.069 0.069 0.069
NaIBBS
293 303 313 323 333 343 353 363
180.5 182.5 181.5 178.5 183.5 186.7 195.7 210.7
0.055 0.055 0.055 0.055 0.055 0.055 0.055 0.055
NaCS
293 303 313 323 333 343 353 363
68.5 83.5 102.5 120.5 137.5 155.5 150.5 151.5
0.045 0.045 0.045 0.045 0.045 0.045 0.045 0.045
NaPTSA
293 303 313 323 333 343 353 363
5.35 5.80 5.50 5.90 6.60 6.55 6.40 6.62
0.014 0.014 0.014 0.014 0.014 0.014 0.014 0.014
PTSA
293 303 313 323 333 343 353 363
5.33 5.75 5.80 5.90 6.10 6.45 6.30 6.62
0.014 0.014 0.014 0.014 0.014 0.014 0.014 0.014
hydrotrope
(1)
The aggregation constant Kn is related to the dimerization constant of the hydrotrope (K2) as Kn ) K2/n. The total concentration of the hydrotrope (Cs) and monomer concentration, [H1], can be related by the equation ∞
Cs )
∑ n[Hn]
(2)
n)1
which reduces to
Cs ) [H1]{2 exp(K2[H1]) - 1}
(3)
The hydrotrope aggregates dissolve the organic solute presumably by trapping it within the aggregate. The association of solute with a hydrotrope aggregate of aggregation number n can be represented by the mass action equilibrium, as if it were co-aggregating with the hydrotrope aggregate
HnSm-1 + S1 S HnSm
(4)
A hydrotrope n-mer (Hn), therefore, can have a population of different solute concentrations, which, in turn, can be related to the hydrotrope-solute interaction parameter, Ksm, through the equation
[HnSm] ) Ksm[HnSm-1][S1]
(5)
A solute molecule residing between the hydrotrope molecules reduces the electrostatic repulsion between the charged groups of the hydrotrope molecules, effectively tightening the structure and providing an additional geometrical constraint to the incorporation of more solute molecules into the same aggregate. The total amount of the solute associated with the hydrotrope aggregates, from the hydrotrope dimer onward, is ∞
ST )
n-1
∑ ∑ m[HnSm]
(6)
n)2 m)1
which reduces to
()
ST ) 2
Ks [S ]{exp(K2[H1]) - (1 + K2[H1])} (7) K2 1
where the constant Ks characterizes the interaction between a solute-free hydrotrope n-mer and the first solute molecule and is related to Ksm, i.e., Ksm ) Ks/m.
A higher value of Ks signifies a stronger interaction of the solute with the hydrotrope aggregate.19 The solubility data of diosgenin, only above the MHC of each hydrotrope, were fitted into the model to estimate the parameters, Ks and K2, which represent the solute-hydrotrope and hydrotrope-hydrotrope interactions, respectively (Table 2). Diosgenin-NaNBBS interactions are clearly the strongest, followed by those of diosgenin with NaIBBS, NaCS, NaPTSA, and PTSA, in that order, as shown by the Ks values with the respective hydrotropes. The hydrotrope-hydrotrope association constant (K2), on the other hand, shows a comparatively poor aggregation tendency of these amphiphilic molecules (Table 2). The presence of hydrophobic solute might, however, augment the aggregation tendency of the hydrotropes, as solubilization is akin to co-aggregation. The marked effect of temperature on the solubility of diosgenin above the MHCs is possibly because of changes in the aggregate structures of the hydrotrope. However, in the absence of any information about the aggregation pattern it is difficult to explain the temperature effect. The solubility in NaCS solutions was affected more than that in other hydrotropes. As the temperature was increased from 293 to 363 K, the diosgenin solubility increased 4.3 times in 2.0 mol/dm3 NaCS solutions as compared to a 2.5-times increase in NaNBBS solutions,
5342 Ind. Eng. Chem. Res., Vol. 43, No. 17, 2004
Figure 2. Effect of temperature on the hydrolysis of dioscin in 2.0 kmol/m3 aqueous NaCS solution containing 1.0 kmol/m3 HCl.
even though the absolute solubility of diosgenin was much higher in the latter. Hydrolysis of Dioscin. Acid hydrolysis of dioscin to diosgenin is essential, either before or after its extraction from the plant material, to recover diosgenin. The hydrolysis of dioscin was carried out in aqueous NaCS solutions, to explore the effect of the hydrotrope on the hydrolysis. Dioscin was first extracted into aqueous 2.0 kmol/m3 NaCS solutions at different temperatures and then hydrolyzed to diosgenin in the presence of an acid (1.0 kmol/m3 HCl) in the same solutions. Figure 2 shows the effect of temperature on the hydrolysis in the hydrotrope solutions. The reaction had to be conducted at temperatures above 333 K, as below this temperature, the rate of hydrolysis was very slow even with 2.0 kmol/m3 HCl acid. At 353 K, the hydrolysis of dioscin was complete in 40 min as compared 4 h in the absence of the hydrotrope.20 This time increased substantially at lower temperatures. With 0.75 kmol/m3 HCl acid, the hydrolysis required 2.5 h for completion, whereas with 0.5 kmol/m3 acid, only 70% of dioscin was hydrolyzed in 5 h, as shown in Figure 3. The hydrolysis rate also showed a first-order dependence on the dioscin concentration, and the estimated rate constants (k) of the hydrolytic reaction at different temperatures, i.e., 0.071 min-1 at 353 K and 0.017 min-1 at 333 K, in the presence of 1.0 kmol/m3 HCl indicate the sensitivity of the reaction to temperature. The energy of activation estimated from the plot of ln k vs 1/T (Figure 4) is 69 kJ/mol. The decomposition of diosgenin to diene in a side reaction also depends on the concentration of the acid.21 Higher acid concentrations and longer times both lead to more loss of diosgenin to diene. The acid hydrolysis of dioscin in ethanol and 2.0 kmol/m3 HCl under refluxing conditions has been reported to give 35% decomposition of diosgenin in 2 h, whereas only 6% of diosgenin decomposition has been reported in the acid solution alone in the same time.22,23 With the hydrotrope solutions, because the time of the hydrolytic reaction has been brought down substantially, it shows benefits
Figure 3. Effect of HCl concentration on hydrolysis kinetics of dioscin in 2.0 kmol/m3 aqueous NaCS solution at 353 K. 0, 1.0; 4, 0.75; O, 0.5 mol/dm3.
Figure 4. Temperature dependence of hydrolysis rate constant in 2.0 kmol/m3 NaCS solutions in the presence of 1.0 kmol/m3 HCl.
by increasing the recovery of diosgenin. Figure 5 shows the effect of temperature on the decomposition of diosgenin to diene in aqueous 2.0 kmol/m3 NaCS solutions. There was no diene formation at all up to 75 min but in the next 2 h, 2.5% of the diosgenin decomposed at 343 K whereas 15% of the diosgenin decomposed to diene at 353 K during the same time. This reaction also showed a first-order dependence on the diosgenin concentration. After completion of the hydrolysis, diosgenin precipitated from the aqueous hydrotrope solution upon cooling to 293 K. The highest recovery of diosgenin could be obtained from hydrotrope solutions containing 1.0 kmol/ m3 HCl at 353 K. This condition was maintained for all further extraction studies. Extraction Studies with Hydrotrope Solutions. The recovery process based on the hydrotropic phenomenon involves extraction of dioscin into hydrotrope solutions in the first step followed by its hydrolysis to diosgenin in the same solution. Diosgenin finally can
Ind. Eng. Chem. Res., Vol. 43, No. 17, 2004 5343
Figure 5. Effect of temperature on the decomposition of diosgenin to diene in aqueous 2.0 kmol/m3 NaCS solutions containing 1.0 kmol/m3 of HCl: O, temperature ) 353 K; 0, temperature ) 343 K.
Figure 7. (a) Effect of water soaking on the dioscorea rhizome section. (b) Effect of aqueous NaCS solutions on the section at room temperature of 303 K. (c) Effect of aqueous NaCS solutions on the section at 343 K.
Figure 6. Comparison of dioscin extraction with water and different hydrotropes (2.0 kmol/m3) at 343 K and purity of diosgenin obtained after hydrolysis (solid loading ) 5% w/v, 16mesh size particles, time ) 5 h): percent extraction (white), percent purity (pattern).
be recovered from the solution by either reducing the temperature, diluting the solution, or both. The suspension density of dioscorea rhizomes for the extraction of dioscin was kept to 5% (w/v). At higher loadings of the 16-mesh size particles and at higher temperatures, the suspension became viscous and thick as the particles absorbed the aqueous solution. Increased dissolution of starch in hydrotrope solutions at higher temperatures increases the viscosity of the solution. Both the sampling and filtration were difficult with such suspensions. Even though dioscin is highly water-soluble, plain water was able to extract only 5% of the dioscin from the rhizomes in 5 h at 343 K (Figure 6). Dioscin, which is present mainly in the stellar region, is protected by outer cork cells and cortex, and water cannot easily penetrate into this region.1,5 Aqueous NaCS solutions, however, extracted 80% of dioscin under identical conditions in 3 h (Figure 6). Hydrotropes
are known to disturb lamellar liquid crystal structures of surfactants in aqueous solutions and are thought to have similar effects on the cell membranes in addition to a hydrolytic effect on the cellulosic structures of plant cells.10-12 The extracted dioscin was hydrolyzed at 353 K with HCl (1.0 kmol/m3). The acid hydrolysis also partly hydrolyzes the dissolved starch and, in the process, improved the solution viscosity. The subsequent dilution of the solution by water to bring the hydrotrope concentration below its MHC precipitated diosgenin from the NaCS solutions as a solid at 303 K. Diosgenin recovered from NaCS solutions was of marginally better quality compared to that obtained from solutions of other hydrotropes and was of substantially improved quality compared with the extraction using water as shown in Figure 6. Effect of Hydrotrope on Dioscorea Rhizome Cells. The effect of aqueous NaCS solutions on the dioscorea rhizome section was studied further for the elucidation of the mechanism of dioscin extraction. Even though dioscin is a water-soluble glycoside, it is difficult to extract with water alone. The structural changes in the dioscorea cells upon exposure to hydrotrope solutions were observed under an Olympus polarizing microscope. Figure 7a-c shows that the cells were swollen after being soaked in water. The exposure of the cells to aqueous hydrotrope solutions, however, not
5344 Ind. Eng. Chem. Res., Vol. 43, No. 17, 2004
Figure 8. Effect of particle size on the rate of dioscin extraction using 2.0 kmol/m3 aqueous NaCS solution at 303 K [solid loading ) 5% (w/v), time ) 5 h].
only led to their swelling but also cleared them completely of some of the intracellular materials. The hydrotrope solution disturbs the organized suberin lamella structure of the cells as hypothesized for the extraction of piperine from pepper cells.11 The hydrotrope solution must diffuse through the cell walls and dissolve the material inside the cells, which is not easy with water alone. The effective penetration of the hydrotrope into the cell depends on the nature of the hydrotrope. It was also observed that NaCS was more effective than NaPTSA and NaXS. The rhizome sections when heated in aqueous NaCS solutions at 343 K for 1 h showed no apparent breaking of cell walls, but the cellular material was secreted to a higher extent into the hydrotrope solutions. These observations indicate that the permeabilization of the cell walls followed by diffusion of the aqueous hydrotrope solution into the cells results in the rapid extraction of dioscin. Effect of Particle Size. Figure 8 shows the effect of particle size on the rate of dioscin extraction into 2.0 mol/dm3 aqueous NaCS solutions. Smaller particles, i.e., 22, 36, and 85 mesh, absorbed a significant amount of aqueous solutions and formed a thick paste. This makes processing of the suspension difficult, and extraction with larger particles becomes necessary from the operational point of view. The outer protective layer of the dioscorea tuber is composed of a number of regularly arranged rows of lignified cork cells, usually 6-7 layers, followed by thin-walled cortical parenchymatous tissue.1,5,13 Dioscin is mainly concentrated in the stellar region of the cell. Starch grains are also abundantly present in the stellar region, and this region is surrounded by cortex. As dioscin is concentrated in the inner part of the cell, smaller particle sizes gave marginally increased extraction of dioscin from 62 to 76%. However, this increased extraction is accompanied by a decreased purity of recovered disogenin because of the increased solubilization of other impurities (Figure 9). The dioscin extraction data showed a first-order behavior, and the extraction rate constant was deter-
Figure 9. Effect of particle size on the extraction of dioscin and the purity of the diosgenin obtained after hydrolysis of dioscin in 2.0 kmol/m3 aqueous NaCS solutions (solid loading ) 5%, time ) 5 h): percent extraction (white), percent purity (pattern).
Figure 10. Effect of aqueous NaCS concentration on the percent extraction of dioscin and diosgenin purity obtained after hydrolysis at 353 K (solid loading ) 5%, 16-mesh size; time ) 5 h, temperature ) 303 K): ), percent extraction; 4, purity of diosgenin without dilution; 0, purity of diosgenin with dilution.
mined for different particle sizes. The rate constant increases with decreasing particle size, indicating the major resistance to mass transfer within the particles. Figure 10 shows the effect of the NaCS concentration on the extraction of dioscin and the purity of diosgenin recovered after the hydrolysis and cooling, with and without dilution, of the solution. There is a significant increase in the percentage extraction in a given time with increased hydrotrope concentration. At lower hydrotrope concentrations, significant amounts of starch and tuber pigments were also extracted into the hydro-
Ind. Eng. Chem. Res., Vol. 43, No. 17, 2004 5345
run. At each extraction stage, there was 4% (w/w) loss of the hydrotrope solution with the solid cake. Extraction of some saponifiable materials such as saponins also causes the solution to froth and foam during the process, increasing with the number of cycles. After the hydrolysis of dioscin under acidic conditions at 353 K in the same solution, ∼70% of the diosgenin precipitated as solid product with a purity of >95% upon cooling because of its poor solubility even in NaCS solutions at 293 K. As no chemical reaction takes place between the hydrotrope and diosgenin, the hydrotrope can be recovered for recycle by addition of common salt to the solution by the common ion effect. However, a better approach would be to conduct the extraction of dioscin at higher temperatures and precipitate the product by temperature reduction, as concentrating the hydrotrope solution before recycling might become a controlling factor for industrial application. Conclusion Figure 11. Effect of temperature on the extraction of dioscin using aqueous NaCS (NaCS concentration ) 2.0 mol/dm3, solid loading ) 5% w/v, 16-mesh size particles, time ) 5 h).
tropic solutions, reducing the product purity. At higher hydrotrope concentrations, the increased penetration of hydrotrope into cellular structures led to better dioscin extraction. Also at the higher NaCS concentrations, mainly diosgenin precipitated from the solution after the hydrolysis when the solution was cooled to 303 K without dilution, resulting in its high purity (>95%). However, the yield of recovered diosgenin by simple cooling was low. It is apparent that the other impurities are retained by the hydrotrope solutions. Simultaneous cooling and dilution, however, resulted in increased disogenin yield but reduced purity. It would be necessary to treat the final solution for the removal of impurities, if the hydrotrope solution needed to be recycled for the next extraction step. Figure 11 shows the effect of temperature on the rate of dioscin extraction in the range of 303-343 K with 2.0 mol/dm3 NaCS solution. The rate and extent of the extraction of dioscin increased with incresing temperature. The purity, however, decreased because of increased solubilization of natural pigments, tannins and starch by the hydrotropic solutions at higher temperatures where a greater extent of cell lysis either by dissolution or disorganization might take place. At 343 K, the maximum dioscin extraction (80%) was achieved in 3 h with a purity of 65%. Lower temperature (303 K) gave comparatively less (∼66%) extraction in 5 h but a better purity of 82%. At higher temperatures, a significant amount of starch also dissolves in the hydrotrope solutions and precipitates during the recovery step, so purification of the final product by removing the starch will be necessary. Recyclability of Hydrotrope Solutions. The reusability of hydrotrope solutions in 2.0 mol/dm3 of aqueous NaCS solutions was verified with 5% (w/v) solid loading of 16-mesh size particles each time, as the amount of dioscin (or diosgenin) extracted in a single stage was not sufficient to saturate the hydrotrope solution. The hydrotrope solution was recycled five times, and dioscin was enriched in the same hydrotrope solution, each time charging fresh roots for the extraction. The samples were withdrawn frequently for analysis. The degree of extraction of dioscin remained almost constant (6663%) for four runs and decreased to 57% in the fifth
The extraction of dioscin from the dioscorea rhizome has been facilitated by the use of aqueous hydrotrope solutions. Permeabilization of the plant cells followed by diffusion of the aqueous hydrotrope solution into the dioscorea rhizome is apparently responsible for the extraction of dioscin. The simplicity of the extraction method, coupled with the easy recovery of the product, makes this process promising. Complete hydrolysis of dioscin to diosgenin was achieved within 40 min with only 1.0 mol/dm3 HCl as compared to 4 h in commercial processes, using 3-4 mol/dm3 HCl, which makes the process potentially attractive. The decomposition of diosgenin to diene, which takes place during the hydrolysis of dioscin, is almost absent in aqueous hydrotropic solutions in short-time runs, giving an added benefit. The purity of diosgenin obtained is very high (>95%) when it precipitates from the aqueous hydrotrope solutions by the temperature effect. Acknowledgment The authors acknowledge financial support from the Department of Science and Technology (DST) of the Government of India to this work. Literature Cited (1) Evans, W. C. Saponins, cardioactive drugs and other steroids. In Trease & Evans Pharmacognosy, 15th ed.; Green, E., Ed.; W. B. Saunders (Harcourt Publishers Ltd.): New York, 2002; p 289. (2) Aradhana, M.; Rao, A. C.; Kale, R. K. Diosgenin a Growth Stimulator of Mammary Gland of Ovarietomized Mouse. Ind. J. Exp. Biol. 1992, 30, 367. (3) Roman, I. D.; Thewles, A.; Coleman, R. Fractionation of Livers following Diosgenin Treatment to Elevate Billary Cholesterol. Biochem. Biophys. Acta 1995, 1255, 77. (4) Sauvaire, Y.; Ribes, G.; Baccou, J. C.; Loubatieeres Mariani, M. M. Implication of Steroid Saponins and Sapogenins in the Hypocholestromic effect of Fenugreek. Lipids 1991, 26, 191. (5) Kokate, C. K.; Purohit, A. P.; Gokhale, S. B. Pharmacognosy, 12th ed.; Nirali Prakashan: Pune, India, 1999; p 174 (6) Applezwig, N. Steroid Drugs; McGraw-Hill: New York, 1962; p 46. (7) Sucrow, W.; Winkler, D. Diosgenin Saponins from Dioscorea floribunda. Phytochemistry 1975, 14, 539. (8) Liu, B.; Lockwood, B. G.; Gifford, H. A. Supercritical Fluid Extraction of Diosgenin from Tubers of Dioscorea nipponica. J. Chromatogr. 1995, 690, 250. (9) Thakkar, V, J.; Saoji, A. N.; Deshmukh, V. K. Effect of Ultrasonic Waves on Extraction of Active Constituents of Some Crude Drugs. Indian J. Pharmacol. 1974, 36, 136.
5346 Ind. Eng. Chem. Res., Vol. 43, No. 17, 2004 (10) Dandekar, D. V.; Gaikar, V. G. Hydrotropic Extraction of Curcuminoids from Turmeric. Sep. Sci. Technol. 2003, 38 (5), 1185. (11) Raman, G.; Gaikar, V. G. Extraction of Piperine from Piper nigrum (Black pepper) by Hydrotropic Solubilization. Ind. Eng. Chem. Res. 2002, 41, 2966. (12) Raman, G.; Gaikar, V. G. Hydrotropic Solubilization of Boswellic Acids from Boswellia serrata Resin. Langmuir 2003, 19, 8026. (13) Indian Herbal Pharmacopoeia; Regional Research Laboratory, CSIR, Jammu and Indian Drugs Manufacture Association: Mumbai, India, 1999; Vol. 2, p 44. (14) Sanchez, G. L.; Medina, J. C.; Soto, R. R. Spectrphotometric Determination of Diosgenin in Dioscorea composita Following Thin Layer Chromatography. Analyst 1972, 97, 973. (15) Gaikar, V. G.; Sharma, M. M. Separations with Hydrotropes. Sep. Technol. 1993, 3, 3. (16) Balasubramanium, D.; Srinivas, V.; Gaikar, V. G.; Sharma, M. M. Aggregation Behavior of Hydrotropic Compounds in Aqueous Solution. J. Phys. Chem. 1989, 93, 3865. (17) Bhat, M. B.; Gaikar, V. G. Characterization of Interaction between Butyl Benzene Sulfonates and Cetyl Trimethylammonium Bromide in a Mixed Aggregate Systems. Langmuir 1999, 15, 4740.
(18) Bhat, M. B.; Gaikar, V. G.; Characterization of Interaction between Butyl Benzene Sulfonates and Cetyl Pyridinium Chloride in a Mixed Aggregate Systems. Langmuir 2000, 16, 1580. (19) Gaikar, V. G.; Phatak, P. V. Selective Solubilization of Isomers in Hydrotrope Solutions: o-/p-Chlorobenzoic Acids and o-/p-Nitroanilines. Sep. Sci. Technol. 1999, 34 (3), 439. (20) Rothrock, J. W.; Hammes, P. A.; McAleer, W. J. Isolation of Diosgenin by Hydrolysis of Saponin. Ind. Eng. Chem. 1957, 49 (2), 186. (21) Yamuchi, T. Saponins of Japanese Dioscoreaceae IX. Hydrolysis of Diosgenin Glycosides. Chem. Pharm. Bull. 1959, 7, 343 (Chem. Abstr. 1960, 54, 24855h). (22) Peal, J. W. Dehydration of Diosgenin During the Acid Hydrolysis of Dioscorea Saponins. Chem. Ind. 1957, 44, 1451. (23) Blunden, G.; Hardman, R.; Morrison, J. C. Quantitative Estimation of Diosgenin in Dioscorea Tubers by Densitometeric Thin Layer Chromatography. J. Pharm. Sci. 1967, 56, 948.
Received for review August 25, 2003 Revised manuscript received March 31, 2004 Accepted May 23, 2004 IE034091V
