Hydrotropic Extraction Process for Recovery of Forskolin from

Ind. Eng. Chem. Res. 2009, 48, 8083–8090

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Hydrotropic Extraction Process for Recovery of Forskolin from Coleus Forskohlii Roots Sanjay P. Mishra and Vilas G. Gaikar* Institute of Chemical Technology, UniVersity of Mumbai, Matunga, Mumbai, 400019, India

A simple and rapid method based on hydrotropic solubilization is developed for isolation of forskolin from coleus forskohlii roots. The plant cells are permeabilized by aqueous hydrotrope solutions followed by extraction and solubilization of forskolin into the hydrotrope solutions of alkyl benzene sulfonates and carboxylates. The solubility of forskolin is increased by 350 times in the hydrotropic solutions and it is possible to recover 85% pure forskolin from the hydrotropic solutions by simple dilution with water. The purity of the recovered forskolin decreased from 85% to 70% on decreasing particle size of the roots. Nearly 80% of the forskolin having 50% purity was recovered from the coleus roots using 2.0 mol/dm3 aqueous sodium cumene sulfonate (Na-CS) solutions at 363 K. Na-CS showed the most efficient solubilization of forskolin from the Coleus roots among all the other hydrotropes. Introduction Forskolin (Figure 1), a diterpene compound, is present in Coleus forskohlii roots as a major constituent along with deacetylforskolin, 9-deoxyforskolin, and 1,9- deoxyforskolin as the minor constituents.1,2 Forskolin shows positive effects against asthma, glaucoma, hypertension, hair loss, cancer, and obesity.3-5 These multifaceted pharmacological activities of forskolin are mainly due to its role as an activator of adenylate cyclase. Although a total synthesis of forskolin has been achieved, it is not yet competitive with the natural product.6 Conventionally, the isolation of forskolin involves extraction by alcohol or mixture of different solvents from pulverized roots followed by its purification by column chromatography or crystallization.7-9 Majeed et al.7 have described a process in which forskolin is extracted from the pulverized Coleus roots with alcohol or a mixture of solvents. The crude forskolin recovered was further purified by crystallization using another mixture of solvents on the basis of its solubility in the solvents. The recovery process for forskolin developed by Tandon et al.8 comprises the extraction of pulverized roots by using dichloroethane. The solid residue recovered from the extract was separated into different fractions by chromatography on silica gel. The impure forskolin fraction was further crystallized to isolate pure forskolin. Recently, Saleem et al.9 have reported a similar process for the isolation of pure forskolin. The pulverized roots were extracted with toluene and the product recovered was purified by charcoal column chromatography. The residue isolated after chromatography separation was purified and crystallized using different solvent mixtures to recover pure forskolin. The use of large volumes of volatile and inflammable solvents and solid handling in large quantities make these processes labor-intensive and environment unfriendly. Development of alternative extraction techniques with better selectivity and efficiency for the natural product are, therefore, highly desirable. Supercritical extraction using CO2 has been gaining importance for extraction of natural products mainly because of the major advantage of getting a product completely free from residual solvent. But most polar components show a limited solubility in pure supercritical CO2 and many times a polar entrainer, usually ethanol, is added to modify the solvent * To whom correspondence should be addressed. E-mail: v.g. [email protected]. Fax: 91-0 22- 24145614.

characteristics under supercritical conditions. Another major limitation of supercritical conditions is the need of high pressure equipment required to maintain the supercritical pressure of the solvent which adds significantly to the cost of separation. In the recent years, we have developed an alternative extraction process using aqueous solutions of hydrotropes for the recovery of naturally occurring secondary metabolites such as curcuminoids from turmeric,10 piperine from black pepper,11 boswellic acids from Boswellia serrata resins,12 diosgenin from Dioscorea rhizome,13 and andrographolide from Andrographis paniculata.14 Hydrotropes are highly water-soluble small molecular weight organic salts and show a strong concentration dependence for their ability to dissolve other organic compounds in aqueous solutions. Hydrotropes show their solubilization capacity for organic compounds above a characteristic minimum hydrotrope concentration (MHC). Thus, if, a concentrated hydrotrope solution, loaded with the dissolved solute, is diluted below the MHC of the hydrotrope, a major amount of the dissolved solute separates out as a different solid/liquid phase, giving an easy method of the solute recovery even at ambient conditions. The hydrotropic solutions can be recycled as no chemical reaction takes place of the hydrotope with the product.10-14 Hydrotropes, particularly carboxylates such as Na-salicylate, have been extensively investigated as drug solubilizers and could be ideal candidates for such extractions.15-17 Moreover, hydrotropes are readily biodegradable under aerobic conditions.18 Because of their high solubility in water, trace amounts, if any, of the hydrotropes from the product can be water washed. We explore here the extraction of forskolin and its recovery in better purity using aqueous hydrotropic solutions unlike multistep purification processes involving multiple organic solvents/ mixtures. This process could also be an alternative to super-

Figure 1. Structure of forskolin.

10.1021/ie801728d CCC: $40.75  2009 American Chemical Society Published on Web 04/07/2009

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Ind. Eng. Chem. Res., Vol. 48, No. 17, 2009

critical fluid extraction method as it uses ambient conditions of pressure and temperature to achieve the extraction and can be operated at any scale. On the other hand, if the basic extraction of the active materials along with other compounds is conducted using hydrotrope solutions on a large scale, a much smaller unit of supercritical extraction could be useful for further purification of the extract recovered from aqueous hydrotrope solutions. Materials and Methods All the solvents and chemicals used were of analytical grade. Dried roots of Coleus forskohlii (Lamiaceae) were obtained from Godavari Sugar Mills Ltd., Mumbai, India. The raw material was first pulverized, and the dry powder was separated by mechanical sieving into different average particle size batches, that is, 2.8-3.0, 0.8-1.0, 0.5- 0.7, 0.25-0.4, and 0.1-0.2 mm, respectively. The hydrotropes, sodium salts of cumene sulfonate (Na-CS) and p-toluene sulfonate (Na-PTS), were purchased from Navdeep Chemicals Ltd., Mumbai, and were recrystallized from methanol and dried before use. Sodium salicylate (Na-Sal) was obtained from Swastik Pharmaceuticals, Mumbai. Pure forskolin was isolated using a reported procedure7-9 and purified by crystallization and used as the standard. Analytical Methods. The analysis was performed using high performance liquid chromatography (HPLC) with Hi-QSil-C18 column of length 250 mm. The column was initially rinsed with methanol for 30 min and then equilibrated with acetonitrile/ water (50: 50) mobile phase. The column was mounted on a Jasco Pu-2080 plus HPLC chromatograph equipped with 20 µL loop injector and photodiode analyzer detector (MD-2010). The mobile phase flow rate was maintained at 1.0 cm3/min and the detection wavelength was 210 nm. The analysis was isocratic and was carried out for 20 min.19 The retention time of forskolin was ∼14.5 min. Solubility Studies. The solubility measurements of forskolin in hydrotrope solutions experiments were carried out in a cylindrical glass vessel of maximum volume 50 cm3 equipped with a six bladed turbine impeller (2 cm i.d.). This entire assembly was kept in a constant temperature water bath of accuracy (0.1 °C during the solubility studies. The experiments were carried out by suspending pure forskolin in aqueous hydrotrope solutions of concentration range 0.05-2.0 mol/dm3. The solution was equilibrated with an excess amount of pure forskolin for 4 h under vigorous stirring at constant temperatures ranging from 20 to 90 °C to attain the equilibrium. After 4 h, the stirring was stopped and the solution was kept still for 15 min for the solid particles to settle. A suitably warmed pipet was used to withdraw the clear upper portion of the solution with the tip protected by a microfilter. This sample was analyzed to determine forskolin solubilized into the hydrotrope solutions. Kinetics of Extraction. The extraction of forskolin from Coleus forskohlii roots of selected particle size was conducted in a fully baffled cylindrical glass vessel (100 cm3) equipped with a six- bladed turbine impeller (2 cm i.d.). Pulverized roots of different average particle size were suspended in aqueous hydrotrope solutions of concentration up to 2.0 mol/dm3. The suspension was agitated vigorously at 1200 rpm for a period of 3 h at which external mass transfer resistance can be considered insignificant. Samples were withdrawn after definite time intervals and analyzed for the forskolin content. After the extraction was conducted for a known time period, the solution was subsequently filtered under vacuum. A clear brown color solution was obtained as filtrate, while the insoluble sticky solid portion was collected as residue. The residue was washed with 10 cm3 of the fresh hydrotrope solution of the same concentra-

Figure 2. Effect of temperature on solubility of forskolin in aqueous NaSal solutions.

tion to remove any extract adhering to it. The filtrate was diluted with water to the respective minimum hydrotrope concentration (MHC) of the hydrotrope. Solid brown color crystals of forskolin that precipitated out from the hydrotrope solutions were isolated by centrifugation or filtration. The precipitated forskolin was dried, weighed, and analyzed for purity.19 The decomposition of forskolin was separately studied with pure samples as well as in 2.0 mol/dm3 aqueous Na-CS solutions at 90 °C for 5 h. During this period, samples from the solutions were withdrawn and analyzed for the forskolin content. The total forskolin content in the raw material was determined separately by continuous Soxhlet extraction with methanol. The raw material was defatted using petroleum ether (bp 40-60 °C) prior to the extraction for 18 h. The extraction was then carried out for 24 h and the total forskolin content of the coleus roots was estimated to be ∼1.5% (w/w). Results and Discussion To evaluate the hydrotropic solubilization for the extraction of forskolin from the Coleus roots, the knowledge of its solubility in different hydrotrope solutions and at different concentrations of each hydrotrope is necessary. Figures 2-4 show the solubility of forskolin in different hydrotrope solutions, viz., Na-CS, Na-PTS, and Na-Sal, as a function of hydrotrope concentration and temperature. In all the three hydrotrope solutions, the solubility of forskolin increases with the hydrotrope concentration and also with temperature. The maximum increase was observed in 2.0 mol/dm3 Na-CS solutions. The forskolin solubility varies in the range 0.009-0.048 g/dm3 in pure water with the temperature increase from 20 to 90 °C. Hydrotropic solubilization has been claimed to be a collective molecular phenomenon, possibly occurring by the intercalation or co-aggregation of a solute with the hydrotrope aggregates


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well as the functional group(s) attached to the carbon skeleton of the solute as it would govern the intercalation of the solute between the hydrotrope molecules. The solubilization of a solute is influenced by its hydrophobic part and also the chain length of an alkyl group of a hydrotrope. A modified chemical association model of hydrotropic solubilization is used to represent the solubilization of forskolin in hydrotrope solutions.24,25 The model considers stepwise aggregation of the hydrotrope molecules and solubilization of the solute via coaggregation with the hydrotropic aggregates. The self-aggregation of a hydrotrope is favored by the hydrophobic effect which is governed by its hydrocarbon structure and is opposed by the electrostatic repulsion between the charged head groups giving rise to an optimum aggregation number (m) of the self-assemblies of the hydrotrope. The model relates aggregation constant (Kn) of formation of an n-mer to the dimerization constant of the hydrotrope (K2) as Kn ) mK2/n where “m” is an optimum size of the aggregate. This equation further suggests that the association constant for the formation of an n-mer increases if the aggregation number is smaller than the optimum size, otherwise it decreases. For instance, if m > n then Kn > K2, while if m < n then Kn < K2.25 The total concentration of the hydrotrope (Cs) and monomer concentration [H1] can then be related by eq 1:25 Figure 3. Effect of temperature on solubility of forskolin in aqueous NaPTS solutions.

[H1] m - 1 + 2 exp(K2m[H1]) (1) m 2 A solute molecule can reside between the hydrotrope molecules and reduce the electrostatic repulsion between the charged groups of the hydrotrope molecules, effectively compacting the aggregate structure and providing geometrical constraint to the incorporation of more solute molecules into the same aggregate. Since most solubility studies with hydrotrope solutions show a sigmoidal nature of the solubility curve with hydrotrope concentration, it is appropriate to assume a finite capacity of a hydrotrope aggregate to solubilize the solute. Considering that the association constant for incorporation of a solute molecule into an n-mer of hydrotrope decreases with every new addition of a solute molecule, a hydrotrope n-mer is assumed to take up a maximum of n-1 solute molecules. The total amount of the solute associated with the hydrotrope aggregates, under the assumption that hydrotrope aggregatesolute association constant decreases with increase in number of solute molecules (j) in the coaggregate (Knj) Ks/j), is given by25 Cs ) 2

and the self-aggregation of hydrotrope molecules in aqueous solutions is considered to be a prerequisite for the enhanced solubility of the solute.20-23 Hydrotrope, above a MHC, is expected to form organized loose nanoassemblies with distinct hydrophobic regions where the solute can be solubilized. The solute molecules may also take part in the aggregation process of the hydrotrope, thereby, forming coaggregates with the hydrotrope molecules in the aqueous solutions. The formation of a stable coaggregate depends on the molecular structure as

)

]

Ks [S1] [exp (mK2[H1]) - (1 + K2[H1])] (2) K2 m2 The constant Ks characterizes the interaction between a solutefree hydrotrope aggregate and the first solute molecule and is taken as independent of the aggregation number of the hydrotrope aggregate. A higher value of Ks signifies stronger interaction of the solute with the hydrotrope aggregate. The solubility data of forskolin, above the MHC of each hydrotrope, was fitted into the above modified association model. The equations are nonlinear and a nonlinear least-squares method had to be adopted. Equation 1 can be inverted into a polynomial where the monomer concentration [H1] can be obtained in terms of total hydrotrope concentration (Cs). This concentration can be substituted into eq 2 to estimate the relevant parameters such as Ks, K2, and m, which represent the solute-hydrotrope, hydrotrope-hydrotrope interactions and optimum aggregation number, respectively. Table 1 shows that forskolin-Na-CS interactions are clearly the strongest followed by those of forskolin with other hydroST ) 2

Figure 4. Effect of temperature on solubility of forskolin in aqueous NaCS solutions.

()

[(

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Table 1. Hydrotrope-Solute (Ks), Hydrotrope-Hydrotrope (K2) Association Constants and Optimum Aggregation Number of Hydrotrope (m) hydrotrope

T (°C)

K2 dm3/mol

Ks dm3/mol

m

Na-CS

20 30 40 50 60 70 80 90

0.129 0.130 0.130 0.137 0.134 0.135 0.135 0.136

230.0 214.7 211.0 224.0 235.7 231.4 236.0 250.9

21

Na-PTS

20 30 40 50 60 70 80 90

0.081 0.081 0.080 0.081 0.081 0.081 0.080 0.081

157.2 150.9 142.9 142.9 141.5 140.4 140.3 146.3

19

Na-Sal

20 30 40 50 60 70 80 90

0.078 0.078 0.078 0.078 0.078 0.078 0.078 0.078

120.0 127.4 126.9 127.6 120.9 115.3 121.9 124.2

18

tropes Na-PTS and Na-Sal, in that order, as shown by the Ks values with the respective hydrotropes. The Ks value for NaCS is 230 ( 20 dm3 mol-1 while those are 145 ( 8.5 and 123 ( 6.0 dm3 mol-1 for Na-PTS and Na-Sal, respectively. The hydrotrope-hydrotrope association constant (K2), on the other hand, shows comparatively very low values thereby indicating a poor aggregation tendency of these amphiphilic molecules among themselves (Table 1). The optimum aggregation number of the respective hydrotropes was also estimated by fitting the solubility data in eq 2. Na-CS has an optimum aggregation number (m) of 21 while Na-PTS and Na-Sal show m equivalent to 19 and 18, respectively. The values of K2 and m do not change significantly with temperature for the respective hydrotropes. The optimum aggregation numbers obtained for Na-CS, Na-PTS, and Na-Sal are comparable to that obtained for structurally similar hydrotrope sodium butyl benzene sulfonate (Na-NBBS) (ca. 35-40) by small angle neutron scattering experiments.26 Different hydrotropes having different characteristic MHCs show different extents of solubilization.10-14 The solubility data show that the forskolin solubility increased with the increase in the concentration of hydrotrope as well as with temperature. The data also show that alkyl chain length of the hydrotrope and functional group attached to the aromatic ring influence the solubilization process. This indicates that the hydrophobic part of a hydrotrope is important in the solubilization of a solute in aqueous hydrotrope solutions. The solubility of forskolin in aqueous Na-CS solutions (2.0 mol/dm3) was enhanced to the extent of 320 times its solubility in pure water at 30 °C. The solubility of forskolin in water is ∼2.5 mg/dm3 at the same temperature. For Na-CS solutions, the increase in the solubility was more significant beyond 1 mol/dm3. With respect to the MHC of Na-CS (∼0.14 mol/dm3), the solubility increases only 90 times up to 1.0 mol/dm3 as compared to 280 times in 2.0 mol/dm3 at 20 °C. The temperature effect on the solubility of forskolin above the MHC of the hydrotropes is probably because of modified aggregate structures of the hydrotropes at higher temperatures.

However, in the absence of any information about the aggregation pattern at higher temperatures it is difficult to explain the temperature effect. The increased solubility of forskolin in hydrotrope solutions with an increase in temperature does not lead to any significant change in the value of Ks. The solubility in aqueous Na-CS solutions was affected the most by the temperature change as compared to that with other hydrotrope solutions. Increasing the temperature from 20 to 90 °C, the forskolin solubility further increased 5 times in aqueous NaCS solutions at 2.0 mol/dm3. As the solubility of forskolin is higher in the case of Na-CS as compared to other hydrotropes, it was selected to conduct further extraction experiments. Extraction Studies with Hydrotrope Solution. Solid suspension density of pulverized Coleus roots for the extraction process was kept at 5% (w/v) in the aqueous hydrotrope solution. A higher solid loading of the powdered roots in the hydrotrope solutions should give a more concentrated forskolin solutions but at the same time higher suspension loading makes the suspension viscous and thick due to absorption of hydrotrope solutions into the solid particles, The stirring of this pseudoplastic slurry makes the extraction process inefficient and the sampling as well as subsequent filtration steps more difficult. To mathematically characterize the extraction process, the extraction data of forskolin was fitted in a mass transfer model for the extraction of active material from a solid matrix as given by eq 327 1 ∂ ∂C(t, x) ∂C(t, x) ) D φ-1 xφ-1 ∂t ∂x ∂x x

(

)

(3)

where t is the time, x is the radial distance in the direction of material transfer in the particle, and φ is the geometric shape factor of the particle. A second order finite difference method was used to discretize the relevant differential equations to solve by the Crank-Nicolson method. Since the extraction experiments were conducted under vigorous agitation the external mass transfer resistance can be safely neglected in the analysis. The extraction kinetics then will be mainly governed by intraparticle mass transfer processes. The internal diffusion coefficient of forskolin within the solid matrix was estimated by the regression analysis of the experimental extraction kinetic data. Figure 5 shows that the experimental values are well represented by the mass transfer model. Diffusion coefficients of forskolin using different hydrotropes for extraction and at different hydrotrope concentrations were estimated from the same model (see later). Hydrotropes have been reported earlier to modify the permeability of plant cells probably by disorganization of the molecules in the cell membrane as well as by hydrolytic effect on the cellulosic structures. This can lead to cell structure lysis to a greater extent at higher temperatures and, therefore, to increased extraction rates of forskolin. Effect of Particle Size on Extraction. Figure 5 shows the effect of particle size on the forskolin extraction from the coleus roots into 2.0 mol/dm3 aqueous Na-CS solutions. Smaller size particles, that is, 0.5-0.7, 0.25-0.4, and 0.1-0.2 mm, absorbed a significant amount of the hydrotrope solutions and formed a thick paste. This makes processing of the suspension difficult and extraction with bigger size particles was necessary from the operational point of view. The rate of extraction depends on how easily the hydrotrope solution penetrates the biomatrix and enables the solubilization of forskolin. The diffusion constant increases, for instance, from 5.22 × 10-15 to 7.11 × 10-15 m2 sec-1, with decrease in the particle size indicating that the major resistance to the mass transfer lies within the particles. The particle size of the raw material had no significant effect


Figure 5. Effect of particle size on the rate of extraction of forskolin using 2.0 mol/dm3 aqueous Na-CS solutions at 30 °C (solid loading, 5% (w/v); time, 3 h).

Figure 6. Effect of particle size on the % extraction and % purity of forskolin: % extraction, clear bar; % purity, shaded bar.

on forskolin extraction below 0.8-1 mm. Figure 6 shows % extraction in 3 h and % purity of forskolin recovered from the hydrotropic solutions. The results indicate that % purity of recovered forskolin decreases with the decrease in the particle size from 85% to 70%. Thus the extraction studies have to be conducted with bigger size particles to recover active material in a purer form.

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Figure 7. Comparison of extraction extent and purity of forskolin isolated from Coleus forskohlii roots with methanol and different hydrotropes: % extraction, clear bar; % purity, shaded bar.

Comparison of Extraction Using Methanol with Different Hydrotrope Solutions. Figure 7 shows the comparison of extents of extraction of forskolin from Coleus roots with methanol and different hydrotropes. The extraction time was maintained the same at 3 h for all runs. The solid suspension density of pulverized roots (0.8-1 mm) was maintained at 5% (w/w). Figure 7 also shows the purity of forskolin recovered from methanol and different hydrotropes. The highest purity of 85% with maximum extraction of 70% was achieved with aqueous Na-CS solutions as compared to methanol which gave 75% extraction with purity of only ∼8%. Forskolin crystallized out as solid particles from the aqueous solutions when diluted with water. The forskolin of highest purity was recovered from the hydrotrope solutions, probably because of the ability of the hydrotrope solutions to retain the impurities such as pigments, oleoresins, starc, and other water-soluble components. The typical high performance liquid chromatography (HPLC) chromatograms of standard forskolin along with the extract using methanol and hydrotrope solutions are shown in Figure 8. The extracts obtained from aq hydrotrope solutions are definitely purer than that obtained from methanol. Extraction studies with Different Hydrotropic Solutions. Figure 9 shows the comparison of forskolin extraction with different hydrotrope solutions of concentration of 2.0 mol/dm3 at 30 °C. Aqueous Na-CS solutions extracted 70% of forskolin, while Na-PTS and Na-Sal solutions could extract only ∼40% and ∼35%, respectively, in 3 h. The role that the structure of a hydrotrope plays in either solubilizing the cell wall constituents or disruption of the cell wall and/or cell membrane cannot be neglected. Hydrotropes are known to disrupt the lamellar liquid crystal structures of surfactants and also to hydrolyze the cellulosic glycosidic linkage.9-13 The structure of raw materials, such as plant cells, can experience a similar destructive effect of the aqueous hydrotrope solutions. Higher solubility as well

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Figure 8. Comparative HPLC chromatograms of extracted forskolin (mobile phase ) ACN/H2O (50:50), flow rat e ) 1.0 cm3/min, detection wavelength ) 210 nm): (a) standard forskolin; (b) hydrotropic extract; (c) organic solvent extract.

as a greater extent of cell permeabilization of cell structure by Na-CS solutions is mainly responsible for the higher extraction of forskolin by Na-CS as compared to Na-PTS and Na-Sal. Figure 10 shows the extraction of forskolin with aqueous NaCS solutions of different concentrations in the range 0.5 to 2.0 mol/dm3, with 0.8-1 mm pulverized roots maintaining solid loading of 5% (w/v). Figure 10 shows a significant increase in the forskolin extraction with the increased hydrotrope concentration. At lower concentration, for example, at 0.5 mol/dm3, NaCS solutions could extract only 15% of forskolin in 3 h at 30 °C while a 2.0 mol/dm3 hydrotrope solution extracted 70% of the material in the same time period. The extraction was limited to 15% with 0.5 mol/dm3 hydrotrope concentration as the apparent solubility of forskolin was only 0.075 g/dm3. A greater extent of permeabilization of the cell wall as well as higher

solubility of forskolin in concentrated hydrotrope solutions can lead to higher extraction with 2.0 mol/dm3 of Na-CS solutions. Figure 9 also shows the hydrotrope concentration dependence of the diffusion constant. The diffusion constant varies from 3.20 × 10-15 to 6.12 × 10-15 m2 sec-1 with the increase in the hydrotrope concentration. The estimated diffusion coefficients of forskolin at different hydrotrope concentrations indicate that the extraction of forskolin increases almost linearly with the increase in the hydrotrope concentration. It might seem that raising temperature for extraction would be a good way to save time by making the extraction process go faster. However, the problem associated with higher temperature for the extraction process is that it leads to acceleration of all the unwanted side processes. Hence, to carry out the extraction process at higher temperatures it became necessary


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Figure 9. Kinetics of forskolin extraction with different hydrotropes at 30 °C. Figure 11. Effect of temperature on extraction of forskolin with 0.8-1 mm particles using aqueous Na-CS solution (Na-CS concentration, 2.0 mol/ dm3; solid loading, 5% w/v).

Figure 10. Effect of concentration of aqueous Na-CS hydrotrope solutions on extraction of forskolin from 0.8-1 mm particles at 30 °C (Na-CS concentration, 0.5-2.0 mol/dm3; solid loading, 5%; time, 3 h).

to determine the degree of forskolin decomposition, if any, at the stated temperature during the time of operation. It has been observed that forskolin does not undergo any decomposition at 90 °C for the period of 5 h. The results obtained for the pure forskolin are, also, reproducible for the hydrotropic extract. Figure 11 shows the effect of the temperature on the rate of forskolin extraction in the range 303-363 K with 2.0 mol/dm3 Na-CS hydrotrope solutions. The percentage extraction of forskolin increased with the increase in temperature. At higher temperatures, a greater extent of cell structure lysis as well as higher solubility of forskolin in the hydrotrope solution makes higher rates of extraction possible. 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 hydrolytic effect on the cellulosic structures of plant cells.7-10 Forskolin is a terpenoid, mainly situated in cytoplasmic vesicles of roots.27 The root is mainly divided into outermost part as cork cells followed by cortex, cambium, and xylem. Cork consists of polygonal cells. Yellowish to brown masses are found in the cells of cork, cortex, medullary rays, and xylem. These are identified as cytoplasmic vesicles containing secondary metabolites/terpenoids.27 These cytoplasmic vesicles are generally 5-12 µm in diameter and found attached with the outer wall of the cork cells by a membranous stalk. This nature of roots would offer a small resistance to penetration by hydrotrope molecule before enabling solubilization of the forskolin into hydrotrope solutions. At higher temperatures, an increased rupture of the cellular structure is possible by hydrolysis of cellulose following increased solubilization of solute into the hydrotropic solution. Because of the breakdown as well as solubilization of the cellulose

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polymers from the cell wall, the contribution of the polymer toward the firmness of the cell wall could be reduced. At higher temperatures, the diffusion coefficients are expectedly high, i.e to 27.14 × 10-15 m2 sec-1 at 90 °C as compared to 6.12 × 10-15 m2 sec-1 at 30 °C indicating higher extraction rates. Forskolin extraction of 80% was completed within 30 min at 363 K. At higher temperatures, a greater extent of hydrotrope molecules diffuses into the cell structure. This led to a significant cell structure lysis due to hydrotrope, thereby increasing the extraction of forskolin. But the purity of recovered forskolin decreased from 85% to 50% with the temperature increase from 30 to 90 °C. This decrease in purity was mainly due to extraction of other impurities into the hydrotropic solutions which also crystallize along with forskolin during its recovery by dilution of the extract with water. Conclusion Forskolin, which is sparingly soluble in water, has been extracted from coleus forskohlii roots using aqueous hydrotropic solutions. Aqueous solutions of sodium cumene sulfonate gave the best extraction and efficiency in cell permeabilization followed by solubilization of forskolin. The solubility of forskolin increases by 2 orders of magnitude in the presence of hydrotropes in the aqueous solutions. It was possible to extract 80% of the material within 30 min at 90 °C. The process was optimized with respect to concentration of hydrotrope solution, particle size, and temperature required for the extraction of forskolin. Hydrotropes are nontoxic, biodegradable, and can be recycled as no chemical reaction takes place between the hydrotrope and forskolin. Acknowledgment The authors acknowledge financial support from Swarnajayanti Cell of Department of Science and Technology (DST), Government of India, for this work. Nomenclature Cs ) total concentration of the hydrotrope(moles /dm3) [H1] ) hydrotrope monomer concentration (moles /dm3) K2 ) hydrotrope- hydrotrope interaction constant (dm3/mol) Ks ) solute- hydrotrope interaction constant (dm3/mol) m ) optimum aggregation number S1 ) solubility of andrographolide in water (g/dm3) ST ) increased solubility due to hydrotrope solution (g/dm3) t ) time k ) extraction rate constant b ) maximum extraction achieved at the specified conditions.

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ReceiVed for reView November 13, 2008 ReVised manuscript receiVed March 14, 2009 Accepted March 25, 2009 IE801728D

Hydrotropic Extraction Process for Recovery of Forskolin from

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