Microwave-Assisted Extraction of Forskolin from Coleus Roots and Its

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Ind. Eng. Chem. Res. 2010, 49, 9271–9278

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Microwave-Assisted Extraction of Forskolin from Coleus Roots and Its Purification by Adsorptive Separation Using Functionalized Polymer Designed by Molecular Simulation Leena P. Devendra and Vilas G. Gaikar* Institute of Chemical Technology, Matunga, Mumbai-400 019, India

The extraction of forskolin from Coleus forskohlii roots using organic solvents was facilitated by irradiation of the raw material by microwave and studied for effect of time of irradiation, power level and water soaking of the raw material prior to irradiation on the rate of extraction. Methanol gave 92% extraction of forskolin after 2 min of the irradiation at 160 W under optimized conditions, with simultaneous increase in the rate of extraction. Molecular modeling approach was taken to design a specific adsorbent for selective adsorption of forskolin analogues from the complex extract. The forskolin analogues showed greater affinity toward a diethanolamine-loaded polymer as compared to forskolin. The purification of forskolin by adsorption on diethanolamine substituted polystyrene validated the predictions from molecular simulation. Introduction Forskolin (colforsin) is a diterpene compound (Figure 1) isolated from the roots and stems of Coleus forskohlii Briq.1,2 Apart from forskolin, its analogues, 7-deacetylforskolin, 9-deoxyforskolin, and 1,9-dideoxyforskolin are present in the roots as minor constituents. Forskolin shows positive effects against asthma, glaucoma, hypertension, hair loss, cancer, and obesity.3-5 It is widely used in various biochemical studies related to cAMP and adenyl cyclase pathways6 and also in recent years, is being marketed for promoting lean body mass.6 Although, total synthesis of forskolin is known, it is uneconomical because of structural complexity of the molecule and the synthetic forskolin is reportedly not as effective as that procured from the natural source.7 Several methods are described in literature for extraction, isolation, and purification of forskolin from the roots of Coleus forskohlii. The extraction of forskolin is commonly conducted by refluxing the powdered roots or stems of Coleus forskohlii in multiple extraction steps, with a large quantity of an organic solvent, such as benzene, chloroform, and toluene.8-11 Concentrating the extract to almost dryness, usually by evaporative recovery of the solvent, leads to a sticky viscous material as a product. A longer time of extraction to extract maximum amount of the active material from the raw material adds significantly to energy consumption and loss of volatile solvents apart from a large inventory of inflammable organic solvents. The use of supercritical CO2 (scCO2) in recent years has been the most acceptable method for getting active ingredients of natural products.12,13 However, the supercritical fluid extraction process does not eliminate the use of organic solvents and at the same time requires expensive high pressure equipment with limited capacity. Mishra and Gaikar14 have recently reported an aqueous-solution-based hydrotropic extraction of forskolin from Coleus forskohlii roots. The use of aqueous solvents is attractive, yet the purity of the product remains a matter of concern. Forskolin has a good market potential, and higher percentage extraction with better purity product will always be attractive. In this paper, we report a microwave-assisted process for rapid extraction of forskolin into organic solvents. The exposure of * To whom correspondence should be addressed: E-mail: vg.gaikar@ ictmumbai.edu.in. Tel.: 091-22-3361 1111/2222. Fax: 091-22-33611020.

raw material to microwave either before the extraction or during the extraction has been reported to lead to faster extraction rates of the ingredients from plant materials such as curcuminoids from turmeric,15 piperine from Piper nigrum,16 solanesol from tobacco leaves,17 and artemisin from artemisin annua.18

Figure 1. Structure of forskolin and analogues: (a) forskolin, (b)1,9dideoxyforskolin, (c)7-deacetylforskolin.

10.1021/ie100495u  2010 American Chemical Society Published on Web 09/02/2010

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The active principles of a natural product are usually associated with structurally similar analogues with lower or no activity profiles in the parent source. The reported methods of purification of forskolin from the crude extract of Coleus forskohlii mostly involve column chromatography on silica gel and/or successive crystallizations involving multiple solvents.2,19-21 As separation and purification processes need to be more selective toward the desired component from a complex natural product, there is an increasing demand for developing newer separation techniques that are more specific, yet simple and safe. Adsorptive techniques have a potential for separation of components, particularly when present in trace quantities.22 In recent years, we have extensively worked on adsorption from nonaqueous solutions using functionalized polymers with specific ligands.23-25 Here, we report molecular simulation as a tool to develop an affinity adsorbent selective for forskolin analogues based on its interaction with the target molecules. Materials and Methods All the solvents and chemicals used were of analytical grade and were procured from SD Fine Chemicals, Mumbai, India. Dried roots of Coleus forskohlii were obtained from Godavari Biorefineries Ltd., Mumbai, India. Chloromethylated polystyrene was obtained from Ion Exchange (I) Ltd., Mumbai, India. It is a macro-porous polymer having particle size ranging from 0.42 to 1.2 mm and specific surface area of 34 m2/g, with a polystyrene backbone cross-linked with 7-8% divinylbenzene with 3.2 mequiv of chloride groups. Pure forskolin, which was isolated using a reported procedure and purified by repeated crystallizations,20 was used as a standard. It was also characterized by LC-MS. Analytical Methods. The high performance liquid chromatography (HPLC) analysis was performed using 5 µm Hypersil C-18 column of length 250 mm. The column was equilibrated with an acetonitrile-water (50:50) mixture as mobile phase at a flow rate of 1.0 cm3/min. The column was mounted on a JASCO PU-2080 plus HPLC chromatograph equipped with a 20 µL loop injector and a photodiode detector (MD-2010). The LC-MS analysis was performed on Finnigan LCQ Advantage Max (LCQ Adv max, LCQAD 30000, Thermoelectron Corporation) using a Hypersil C18 column and acetonitrile: water mixture as mobile phase at a flow rate of 0.5 cm3/min followed by MS detection on an electron spray ionization source. Pure and dry nitrogen was used as a sheath gas or neubilzer gas with flow rate of 40 cm3/min and auxiliary nitrogen flow rate was maintained at 18 cm3 /min. The capillary temperature was maintained at 275 °C with the voltage at -420 V and ion spray voltage at 5 KV. Microwave-Assisted Extraction. The raw material (15 g) was irradiated with microwave, after it was evenly and thinly spread over a Petri dish, in a microwave oven for a predetermined time at desired power levels. After the irradiation, the material was used for the extraction by suspending it in 150 cm3 methanol. The raw material was also soaked in water for different time periods before subjecting it to microwave irradiation. The extraction of forskolin was studied in a fully baffled cylindrical glass vessel of capacity 250 cm3 equipped with a four-bladed flat blade turbine impeller (2 cm i.d.). The pulverized roots of Coleus forskohlii (0.8-1.0 mm) were suspended in methanol (150 cm3) with 10% (w/w) solid loading. The suspension was agitated vigorously at 1200 rpm at 30 °C for 2 h. The rate of extraction became independent of the speed of agitation beyond 1000 rpm. Samples were withdrawn from the

solution at definite time intervals for the analysis using a microfilter-fitted pipet. After each extraction run, the crude product was recovered by evaporation of the solvent and analyzed for its purity by HPLC. The percentage extraction of forskolin was estimated on the basis of the total forskolin content of the raw material. Soxhlet extraction was separately carried out with methanol for 48 h to estimate the net forskolin content in the Coleus forskohlii roots to be 1.0% (w/w). Decolorization of Crude Forskolin Extract Using Alumina. The methanolic extract was concentrated by evaporating methanol up to 80% of its original volume. Crude forskolin was crystallized from the concentrated solutions by simultaneous addition of petroleum ether and water in a 1:1 volumetric ratio. Petroleum ether was added to get rid of waxy oleoresinous material and also to crystallize forskolin from the solution as forskolin is insoluble in petroleum ether. Water was added to separate coleus oil from forskolin extract as an emulsion. Forskolin crystallized from the above process was a free-flowing and brown colored powder with 30-35% purity. The crude forskolin was dissolved in acetonitrile and further decolorized by adsorption on an alumina column (30 cm × 1.5 cm) with 15% loading of the crude extract based on the weight of alumina. Elution of the adsorbed materials from the alumina column was also carried out using acetonitrile. All other impurities were retained by the alumina column which did not elute out with acetonitrile. The eluate was further used for recovery of pure forskolin by separation from other two analogues. The product was further subjected to LC-MS analysis so as to characterize forskolin and its analogues. Loading of Diethanolamine on Chloromethylated Polystyrene. Diethanolamine was loaded on chloromethylated polystyrene matrix by suspending the polymer beads in solutions of diethanolamine in methanol. The reaction was carried out at 60 °C for 6 h under stirring conditions in a well-stirred reactor. The solution samples were taken during the course of the reaction and were analyzed for the amine content. The amine loading on the polymer was estimated by checking the residual amine concentration in the solution as determined by titration against 0.1 mol/dm3 aqueous HCl solutions using methyl red indicator. The adsorbent was separated from the suspension by filtration and was washed thoroughly with methanol, vacuumdried, and then characterized by Perkin-Elmer FTIR spectrometer (Spectrum BX-II) in a KBr pellet. The loading of the amine on the polymer takes place with simultaneous formation of HCl. The residual amine in the solution showed replacement of 80% of chloride groups by the amine. The IR spectrum also revealed, however, a small peak corresponding to C-Cl stretching indicating the presence of a small amount of unreacted chloride groups in the final adsorbent. Adsorption of Forskolin and Its Analogues on AmineLoaded Polymer. The mixture of forskolin and its analogues in acetonitrile was used as a feed solution for adsorption on the amine-loaded polymer. The polymer (2 g) was packed in a glass column (14 × 0.8 cm). The top and the bottom zones of the column were packed with glass beads. The feed solution was pumped through the packed adsorption bed by a peristaltic pump with flow rate of 1.0 cm3/min in upward direction. Samples were collected at regular time intervals at the exit of the column for 1.5 h and analyzed by HPLC. Once the column was saturated, as indicated by leakage of forskolin in the effluent of the column, the solution flow was replaced by pure acetonitrile. The adsorbed forskolin on the polymer bed was desorbed with an upward flow of acetonitrile at the rate of 1.0 cm3/min. Samples were also

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Figure 2. Effect of microwave irradiation time on forskolin extraction (power, 160 W; particle size, 0.8-1.0 mm; solvent, methanol; temperature, 30 °C; rpm, 1200) (lines are the fitted values).

collected during the desorption stage and were subjected to HPLC analysis as described above. Results and Discussion Extraction Using Organic Solvents. The choice of a solvent for extraction of forskolin from Coleus forskholi is dictated by the solubility of forskolin in the solvent and its dielectric properties. Methanol, ethyl acetate, and acetone were selected for the extraction studies on the basis of their polarity. The percentage extraction of forskolin was estimated on the basis of total forskolin content of the raw material. The percentage extraction of forskolin was maximum in methanol (75%), intermediate in ethyl acetate (36%), and the least in acetone (14%) in 2 h. For identical loadings of the raw material in the suspension, it was also observed that the percentage of forskolin in the crude product was the lowest (2.7%) in acetone, while it was intermediate with methanol (5%), and maximum with ethyl acetate (8%). This is because of lesser extraction of other polar organic constituents from the raw material into ethyl acetate. However, it is clear that selective extraction of forskolin is not feasible with either of the solvents, and subsequent purification steps are essential to recover forskolin in purer form. Microwave-Assisted Extraction. Our first attempt was to investigate the effect of microwave radiation on the rate of extraction of forskolin into methanol. An enhancement in the rate of extraction was observed after exposure of the roots to microwave at 160 W for 2 min as compared to unirradiated raw material as shown in Figure 2. The striking feature, however, is the improved extent of extraction because of the exposure of the raw material to microwave. About 88% extraction of forskolin was obtained with the irradiated samples as compared to 72% extraction for the unirradiated samples in

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15 min of the extraction period. The subsequent extraction rate was very slow. In 2 h, the extraction reached to 92% for the irradiated samples against 78% for the unirradiated material. The purity of forskolin in the crude product, however, decreased from 5% in the unirradiated material to 3.8% in the extract of the irradiated material. Apart from forskolin, its analogues, that is, 1,9-dideoxyforskolin and 7-deacetylforskolin, were also extracted at faster rates, thereby suggesting an enhanced extraction of all secondary metabolites. In the case of the microwave irradiated samples, some highly polar unidentified oleoresinous organic material was also extracted along with forskolin. The release of additional organic constituents from the raw material because of the rupture of the cellular matrix due to microwave heating can reduce the purity of the extract. The increased rates of extraction of forskolin from irradiated samples are apparently because of facilitated accessibility of the solvent to the forskolin situated in the innermost parts of the cellular structure. Forskolin is a secondary metabolite found in the cells of cork, cortex, medullary rays, and xylem as cytoplasmic vesicles.26,27 Forskolin is thrown out to the outer cork cells to a greater extent and, hence, is expected to have a higher rate of extraction as such even without microwave irradiation. However, the extraction of forskolin located deep in the cells of xylem and medullary rays can be slower because of resistance to the penetration of organic solvent. The dielectric heating of a cellular matrix on microwave irradiation results in the degradation of cellulosic layers of the plant materials at higher temperatures28 and, when subjected to microwave radiation, also increases the permeability of the solvent into the biomatrix which subsequently leads to shorter extraction times. When microwave energy is applied to the raw material, the intracellular material including cellular water coalesces,29 the coalesced material is pushed toward the cell wall, the pressure buildup inside the cell eventually overcomes the mechanical strength of the cell, and the structure explodes.30 The organic solvent can now rapidly diffuse into more open structures and dissolve forskolin. This results in increased mass transfer rates of the intracellular substances as well as improved extent of extraction. A mass transfer model31 for extraction of an active material from a solid matrix was used to mathematically characterize the extraction process of forskolin. The transport of the forskolin within the particle is considered as a diffusion process and is characterized by an effective diffusion coefficient (De). The general equations for solid-liquid diffusion mass transfer are ∂C(t, x) 1 ∂ ∂C(t, x) ) De x ∂t x ∂x ∂x

(

)

(1)

( ∂C(t,∂t x) )

(2)

∂CLt ∂t

(3)

F ) -DeA

F ) VL

A finite difference method was used to discretize the relevant differential equations to solve by the Crank-Nicolson method.32 The diffusion coefficients for extraction of forskolin for all the extraction runs with varying parameters were estimated by the regression analysis with the experimental data. The experimental values are well represented by the given model with a plate shape factor. The De values of forskolin in the solid matrix showed a marginal increase because of microwave irradiation of the raw material from 1.61 × 10-12 m2/sec to 1.71 × 10-12 m2/sec. This increase indeed indicates a few structural changes

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Figure 3. Effect of microwave power on forskolin extraction (time of irradiation, 1 min; particle size, 0.8-1.0 mm; solvent, methanol; temperature, 30 °C; rpm, 1200) (lines are the fitted values).

in the plant cell on exposure to microwave radiation. The De values are still significantly lower than diffusivity values in liquid solutions and thus intraparticle transport seems to be the limiting factor in the large-scale extraction of forskolin. The natural material can have innumerable species, each capable of undergoing reactions of different types, which can lead to loss of the active material. Faster and more complete extraction is definitely, therefore, advantageous. Irradiating the raw material at 160 W showed a significant effect on the percentage extraction of forskolin. However, further increase in the irradiation time, beyond 2 min, did not increase the percentage extraction nor did it show any significant change in the rate of extraction. The microwave power level was, therefore, increased to 360 W, keeping the time of irradiation at 1 min. Figure 3 shows that with increase in microwave power, the percentage extraction of forskolin decreased slightly to 78% as compared to 83% achieved at 160 W after 10 min which indicates thermal degradation of the active material on exposure to microwave at higher power levels. This decrease in the extraction was found to be greater than the variation in the duplicate runs. However, the extraction rate was higher with the raw material irradiated at 360 W. An increase in the microwave power led to decreased extraction of forskolin analogues as well. The percentage of methanol insoluble material was slightly high (85%) as compared to 80% obtained from the material irradiated at 160 W. The increase in the methanol insoluble material indicates degradation of some of the secondary metabolites at higher microwave power levels. The irradiation of the sample for still longer times at the same power level led to complete loss of the material. The rapid heating of the polar organic constituents of the matrix and higher temperatures can cause complete degradation of the active components. Only a dielectric material or solvent with permanent dipole, like moisture, absorbs microwave energy more efficiently. The moisture when heated up inside the plant cells due to microwave effect can generate tremendous pressure on the cell wall when

Figure 4. Effect of time of water soaking on extraction of forskolin (power, 160 W; time of irradiation, 1 min; particle size, 0.8-1.0 mm; solvent, methanol; temperature, 30 °C; rpm, 1200) (lines are the fitted values).

evaporated. The pressure pushes the cell wall from inside, stretching and ultimately rupturing it, which facilitates leaching of the active constituents from the ruptured cells to the surrounding solvent thus improving the percentage extraction. The vacuum-dried raw material after soaking in water for a predetermined time was subjected to the irradiation for 1 min at 160 W. The soaking time of 6 h did not show any significant effect on the percentage extraction (80%) as well as in the extraction rate. However, the 24 h soaking time led to a marginal enhancement in the rate as well as in the final percentage extraction value to 82 (Figure 4). A significant enhancement in the purity of forskolin was observed with the increase in water soaking time as compared to unsoaked and irradiated materials. A 6 h soaking time gave 12% forskolin in the extract whereas the soaking time of 24 h gave 14% forskolin as compared to 3% forskolin in the extract obtained from the unsoaked material. It was also observed that percentage extraction of 1,9-dideoxyforskolin and 7-deacetylforskolin also increased with the increase in soaking time, whereas the percentage extraction of other organic materials did not show any appreciable change. Rapid heating of water leads to faster rupture of the cellular matrix and preferential leaching of polar constituents. The major extraction is over in the first 4-5 min itself. The presence of water might also limit the temperature rise inside the cellular structures, but the increase in pressure could be just enough to break down the cellular matrix. Soaking of the raw material in water for a longer time also showed a significant effect on De values of forskolin as the initial rate of the extraction was found to be higher. The raw material was viewed under scanning electron microscope (Jeol JSM-6380LA), before and after microwave irradiation. Figure 5a shows intact and ordered cellular matrix of the coleus roots before microwave irradiation while Figure 5b shows a more open structure after exposure to the microwave radiation. The cells also appear to be expanded after exposure to the radiation. Expansion causes structural changes in the cell

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Figure 5. SEM images of coleus roots (a) before microwave irradiation and (b) after microwave irradiation.

from within, which in turn leads to pressure buildup inside the cell as result of which cell gets ruptured. Decolorization of Crude Forskolin Extracts Using Alumina. When forskolin extract in acetonitrile was passed over an alumina bed, all brown colored organic impurities of the crude extract were strongly retained by the column. Crude forskolin extract contained 40% of forskolin and its analogues and the remaining 60% was unidentified organic impurities. After decolorization, it was observed that 15% of forskolin, 23% of 7-deacetylforskolin, and 22% of 1,9-dideoxyforskolin from the feed were retained on the column. So based on initial amount of the material taken and the material finally recovered after decolorization, nearly 93.4% of unidentified organic impurities were adsorbed on the alumina column. The light yellow eluate from the column contained 78% (w/w) forskolin, 15% (w/w) of 7-deacetylforskolin and 6% (w/w) of 1,9-dideoxyforskolin, respectively, with only trace amounts of all other impurities. Forskolin and its analogues, being diterpenoids, are neutral molecules, whereas alumina is a highly polar adsorbent. The adsorptive results suggest that the brown-colored organic impurities of the crude forskolin extract are highly polar and hence were strongly retained on the alumina column, whereas forskolin and its analogues being relatively less polar eluted out of the column. No separation of forskolin and its analogues was, however, achieved with the alumina column but the product contained no other impurities. Forskolin and its analogues from the alumina column were further desorbed using 50 cm3 methanol. The colored impurities, on the top portion of the alumina column, remained uneluted even with hot methanol wash and hence nearly 10% of the alumina bed that could not be regenerated had to be compensated with the required amount of fresh alumina in the subsequent experimental runs.

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Molecular Modeling Approach for Engineering an Affinity Adsorbent for Separation of Forskolin and Its Analogues. The final product after removal of colored impurities, contained 1,9-dideoxyforskolin, and 7-deacetylforskolin which differ in terms of number and positions of hydroxyl groups. The hydroxyl groups at first and ninth positions are unique in the forskolin structure. To separate forskolin from its analogues, it was necessary to develop a suitable adsorbent which shows the maximum affinity preferably toward analogues because of their lesser content in the extract. The forskolin analogues are expected to interact with an adsorbent through hydrogen bonding, if the adsorbent was loaded with suitable groups. We decided to load diethanolamine as a ligand on a polystyrene matrix. The amino group was used to anchor the ligand on the polymer matrix while two hydroxyl groups were considered for interaction with the forskolin and its analogues in a differential manner because of relative spatial positions of different hydroxyl groups on the structures of interacting molecules. The ligand is cheaply available, and loading on a polystyrene matrix is a straightforward process. The interaction between the adsorbent and the solute was first studied theoretically by molecular modeling. It may not be possible to predict a priori the adsorption values or the absolute values of the interaction constants with either forskolin or its analogues, but relative adsorption characteristics can be compared to either select an adsorbent for a process or design a new adsorbent for the same. Material Studio 3.2 (MS, Acclerys, USA) was used for the molecular simulation. Each solute and two units of the amine-loaded polystyrene were created separately in a solvent periodic box containing 50 solvent molecules to include the solvation effect and then optimized for intrinsic energy individually using COMPASS (condensed-phase optimized molecular potentials for atomistic simulation studies) as force field and calculation of charges using QEQ. COMPASS is a first high quality ab initio force field to consolidate parameters of organic and inorganic materials and has a broad coverage in covalent molecules including most common organics, small inorganic molecules, and polymers.33 For such molecular systems, the COMPASS force field has been parametrized to predict various properties of molecules in isolation and in condensed phases.34 Both van der Waals and electrostatic interactions were included in the calculation of optimized energy in the Forcite module of MS. The basis of the QEq method is the equilibration of atomic electrostatic potentials with respect to a local charge distribution.35 The interaction studies were also conducted in different solvent media, by simulating the solution with a solvent box of a fixed dielectric constant containing discrete solvent molecules. To simulate the solution of forskolin in a solvent, a cubic simulation box was created with periodic boundary conditions in all directions. A typical periodic box had 50 solvent molecules with size 35 × 35 × 35 Å3. One molecule of forskolin or its analogues and two molecules of repeating unit of the amine-loaded polymer were added into it. The entire system was geometry-optimized for the lowest free energy configuration. The lowest energy configuration was selected as the configuration of a stabilized complex of forskolin with the ligand structure on the polymer. The interaction energy, normalized over per mol of forskolin in kcal/mol, was calculated by subtracting energies of the forskolin and ligand molecules from the energy of the complex. The interaction energy in the solvated state was calculated as follows

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IE ) energycomplex in solvent - (energyforskolin in vacuum + energyligand in vacuum + energysolvent box) (4) The optimized structure of forskolin exhibits intramolecular hydrogen bonding between the adjacent hydroxyl groups at first and ninth positions and between the hydroxyl group at the sixth position and the adjacent acetyl group at seventh position. 1,9Dideoxyforskolin also shows hydrogen bonding between the hydroxyl group at the sixth position and oxygen atom of the acetyl group at the seventh position. 7-Deacetylforskolin exhibits multiple hydrogen bonding among the adjacent hydroxyl groups. The intramolecular hydrogen bonding between different hydroxyl groups and acetyl group on forskolin and its analogues reduces the polar nature of these molecules. Their weaker adsorption on alumina column, therefore, can be explained. 1,9Dideoxyforskolin (energy ) -169 kcal/mol) was found to be more stable with the lowest energy than forskolin (energy ) -103 kcal/mol) and 7-deacetylforskolin (energy ) -39 kcal/ mol). The hydroxyl groups of amine-loaded polystyrene were expected to interact with hydroxyl groups of forskolin or its analogues. A single unit of diethanolamine could not interact with all the hydroxyl groups of the solute molecule simultaneously owing to their stereochemical positions. Considering the positions of various hydroxyl groups in forskolin and its analogues, two units of diethanolamine on the polystyrene structure also were taken on two different sides of the target molecule to study the interaction with a single molecule of forskolin or its analogues. The interaction energy in vacuum was the least for forskolin (-19 kcal/mol), intermediate for 7-deacetylforskolin (-21 kcal/ mol) and maximum for 1,9-dideoxyforskolin (-22 kcal/mol) indicating better interaction of the latter with the ligand molecules. In the case of forskolin, both the hydroxyl groups at first and ninth positions interact with the hydroxyl groups of the amine by hydrogen bonding. The hydroxyl group at the sixth position and acetyl group at the seventh position also show hydrogen-bond formation with the hydroxyl groups of the amine as shown in Figure 6a. In the case of 1,9-dideoxyforskolin, the absence of hydroxyl groups at the first and ninth positions makes the approach of ligand molecules easier for interaction with the carbonyl group at the 10th position as shown in Figure 6b. In case of 7-deacetylforskolin, the four hydroxyl groups are seen forming hydrogen bonds with the hydroxyl groups of the amine (Figure 6c) and thereby showing a greater affinity toward the polymer. In the case of interaction studies in the solvated state in acetonitrile, none of the functional groups of forskolin showed any interaction with the amine-loaded polymer, whereas in the case of 7-deacetylforskolin all the four hydroxyl groups interact with the hydroxyl groups of the amine-loaded polymer. In the case of 1,9-dideoxyforskolin, the acetyl group at seventh position interacts with the hydroxyl groups of the amine-loaded polymer. The interaction energy of 1,9-dideoxyforskolin (-138 kcal/mol) in acetonitrile was found to be maximum as compared to forskolin (-92 kcal/mol) and 7-deacetylforskolin (-131 kcal/ mol), thereby suggesting a greater affinity of the forskolin analogues toward the amine-loaded polymer from acetonitrile solutions. Acetonitrile is a smaller molecule with no hydroxyl groups. Hence, it does not exhibit any hydrogen bonding within itself and neither with forskolin or its analogues. Therefore, forskolin and its analogues are more readily adsorbed on the surface of the adsorbent. In the case of methanol solutions, on the other hand, because of strong intermolecular hydrogen

Figure 6. Interaction of forskolin and its analogues with diethanol amineloaded polymer: (a) forskolin with diethanol amine-loaded polymer, (b) 1,9-dideoxyforskolin with diethanol amine-loaded polymer, (c) 7-deacetylforskolin with diethanol amine-loaded polymer.

bonding of the methanol with hydroxyl groups of forskolin and its analogues, the affinity of the analogues over the amine-loaded polymer was not observed. The optimized structures were further studied for their quantitative structure-activity relationship (QSAR) properties, viz., heat of formation and electrostatic potential surface. In the case of the 1,9-dideoxyforskolin-polymer pair, the heat of formation was less than the sum of their individual heats of formation. The decrease for the 1,9-dideoxyforskolin-polymer pair (-95 kcal/mol) was greater than that for the 7-deacetylforskolin-polymer pair (-91 kcal/mol) and that for forskolinpolymer pair (-84 kcal/mol). A lower decrease in the heat of formation of forskolin indicates that it has a less stable nature and that the formation of such complexes is thermodynamically less feasible. This can explain the preferential affinity of 7-deacetylforskolin and 1,9-dideoxyforskolin toward the amineloaded polymer as compared to that of forskolin. The electrostatic potential surface, which is a measure of electronegativity among various atoms in the molecule, showed that the hydrogen bonding between the forskolin and the diethanolamine-loaded polymer was not very significant as the electronegativity difference between the oxygen and the hydrogen atoms was not very strong, whereas in the case of 1,9-dideoxyforskolin

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Figure 7. (a) Adsorption of forskolin and its analogues over diethanol amineloaded polymer from acetonitrile solutions. (b) Desorption of forskolin and its analogues from diethanol amine-loaded polymer in acetonitrile.

and 7-deacetylforskolin strong electronegativity differences between oxygen atoms and hydrogen atoms involved in hydrogen bonding were observed. (see Supporting Information). These theoretical calculations indicate possibility of separating forskolin from its analogues by selective adsorption of the latter on a diethanolamine-loaded polystyrene matrix from acetonitrile solutions but not from methanol solutions. Adsorption/Desorption of Forskolin and Its Analogues on Diethanol Amine-Loaded Polystyrene from Acetonitrile Solutions. The eluate obtained after decolorization of the crude forskolin extract through a alumina column mainly contained forskolin and its analogues and, therefore, was used as a feed solution for adsorption on the amine-loaded polymer loaded in a column to investigate their adsorption behavior. The dimensionless concentrations (C/C0) of forskolin and its analogues are shown against the number of bed volumes of solution that has been passed through the column in Figure 7a. The uptake by the polymer from the acetonitrile solution was found to be maximum for 1,9-dideoxyforskolin (91%), intermediate for 7-deacetylforskolin (37%), and minimum for forskolin (12.2%) until five bed volumes. These differential uptakes validate the hypothesis from molecular simulation that analogues shall be adsorbed more preferentially than forskolin on the polymer. Gradually, with the passage of six bed volumes, the residual concentrations of forskolin and its analogues became the same in the effluent as that in the feed indicating the saturation of the bed with the solutes. The selective uptake of forskolin analogues by the amine polymer is considered in terms of hydrogen bonding between the hydroxyl groups of the analogues and the hydroxyl groups of the diethanolamine polymer. Forskolin with its hydroxyl groups being intramolecularly hydrogen-bonded is not easily available for interaction with the amine polymer, resulting in its lower uptake. The carbonyl group of 1,9-dideoxyforskolin, however, can freely form a hydrogenbonded complex with hydroxyl groups of the amine polymer resulting in its substantial uptake. The higher uptake of

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7-deacetylforskolin by the amine polymer is due to stronger intermolecular hydrogen bonding with the amine polymer. Figure 7b shows that forskolin gets eluted with acetonitrile at a faster rate as compared to 7-deacetylforskolin which clearly suggests that analogues are having greater affinity toward the amine polymer. 1,9-Dideoxyforskolin was poorly eluted by acetonitrile. These analogues, being strongly adsorbed on the polymer, do not desorb easily as compared to forskolin. During the desorption, the percent purity of forskolin in the eluate and the rate of desorption of forskolin were much higher as compared to its analogues. Almost 90% of forskolin was desorbed within five bed volumes, whereas only 1% of 1,9dideoxyforskolin and 15% of 7-deacetylforskolin were desorbed, respectively, during the same time period. Hence, forskolin with a purity of 94% was obtained in the eluted samples. From the methanolic solutions, however, the uptakes and rates of adsorption of forskolin and its analogues were nearly the same indicating significant effect of the solvent nature on the adsorption characteristics. Methanol is an alcoholic solvent with hydroxyl groups that can have affinity toward the hydroxyl groups of forskolin and its analogues. Hence, there is no significant difference in the rates of adsorption as well as in the percentage adsorption of forskolin and its analogues on the same polymer. Therefore, no separation could be achieved with methanol as the solvent. The forskolin analogues are, however, recovered by passing methanol at 30 °C through the column after adsorption from the acetonitrile solutions. Nearly 74% of 1,9-dideoxyforskolin and 90% of 7-deacetylforskolin was recovered from the affinity adsorption bed. The molecular modeling approach predicted greater affinity of forskolin analogues toward diethanol amine-loaded polystyrene in acetonitrile. Experimental results showed 90 and 37% adsorption of 1,9-dideoxy forskolin and 7-deacetyl forskolin, respectively, as compared to 12% adsorption of forskolin. The experimental data showed a good agreement with the simulation results. Thus molecular simulation can help in designing new adsorbents with desired selectivity and can predict the adsorption profile of various components in a relative sense with more realistic approach by considering the solvent effect. Conclusion Microwave irradiation of raw material, results in the enhancement of rate of extraction of forskolin as well as extent of extraction as compared to extraction using unirradiated raw material. Almost 92% extraction of forskolin was achieved after 2 min of irradiation at 160 W as compared to 78% extraction from unirradiated material. A diethanol amine-loaded polystyrene as functionalized polymer was designed as an adsorbent on the basis of its interaction with forskolin and its analogues by molecular modeling. The molecular simulation also effectively predicted the effect of solvent on selective adsorption by the polymer. Acknowledgment The authors acknowledge financial support from the Department of Science and Technology (DST) of Government of India, to this work. Supporting Information Available: Hydrogen bonding of forskolin and forskolin analogues. This material is available free of charge via the Internet at http://pubs.acs.org. Nomenclature A ) specific area of the solid particle (m2) C ) concentration in solid (kg/kg)

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CL ) concentration in solution (kg/L) De ) diffusion coefficient of the material (m2/sec) VL ) volume of solvent (m3) T ) temperature (°C) t ) time (sec) F ) mass flow, kg/sec

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ReceiVed for reView March 5, 2010 ReVised manuscript receiVed July 29, 2010 Accepted August 15, 2010 IE100495U