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Hydrotropic Solubilization of Boswellic Acids from Boswellia serrata Resin Girija Raman and Vilas G. Gaikar* Institute of Chemical Technology, University of Mumbai, Matunga, Mumbai-400019, India Received April 9, 2003. In Final Form: May 23, 2003 Hydrotropic solubilization of total boswellic acids from Boswellia serrata gum resins has been investigated with aqueous solutions of alkylbenzene sulfonate hydrotropes. Due to their amphiphilic structures, the hydrotropes cooperatively form microassemblies in aqueous solutions which, in turn, are responsible for the solubilization of water-insoluble organic substances. The solubility of boswellic acids was increased by 2 orders of magnitude in the presence of hydrotropes in aqueous solutions. The effect of hydrotrope concentration on the solubility was investigated in detail along with the kinetics of solubilization. The selectivity toward boswellic acids over other terpene compounds was investigated by molecular modeling using the AMBER94 force field.
Introduction Boswellic acids, the pentacyclic and tetracyclic triterpenoids present in the gum resins of Boswellia serrata, show a marked analgesic activity (Figure 1).1 The resin also contains monoterpenes such as R-thujene, diterpenes, triterpenes such as R- and β-amyrins and tetracyclic triterpene acids.2-4 The total acid fraction of pentacyclic triterpene acids is pharmacologically active.5 Conventionally, boswellic acids are solvent extracted from the oleogum resin from the B. serrata tree after defatting.6 Large traces of solvent usually remain in the extract, and the purification steps contribute to higher operating cost and present difficult processing conditions. Structurally, boswellic acids are made up of carbon rings fused together forming the shape of a typical steroid molecule. Bile salts (dihydroxy-5β-cholan-24-oic acids), as a close analogues of sterols, act as solubilizers aiding the fat absorption process in the digestive system by solubilizing lecithin and cholesterol in the intestine.7 Cholesterol is sparingly soluble in water but highly soluble in bile salt solutions.8 Since boswellic acids are very similar to sterols in structure, it is probable that they too will show similar solubilization characteristics. * To whom correspondence should be addressed. Tel: 91-2224145614. Fax: 91-22-4145414. E-mail:
[email protected];
[email protected]. (1) Pardhy, R. S.; Bhattacharyya, S. C. Tetracyclic Triterpene Acids from the Resin of Boswellia serrata Roxb. Ind. J. Chem. 1978, 16, 174175. (2) Pardhy, R. S.; Bhattacharyya, S. C. β-Boswellic acid, Acetyl β-boswellic acid, Acetyl-11-keto-β-boswellic Acid and 11-keto β-boswellic Acid, Four Pentacyclic Triterpene Acids from the Resin of Boswellia serrata Roxb. Ind. J. Chem. 1978, 16B, 176-178. (3) Singh, G. B. Boswellic acids: A new class of anti-inflammatory agents with a novel mode of action. Paper presented at the International Seminar: Traditional Medicine, Calcutta, India, 7-9 November, 1992; pp 81-82. (4) Safayhi, H.; Schweizer, S.; Boden, S. E.; Bayer, E.; Hermann, P. T. A. Workup-Dependent Formation of 5-Lipoxygenase Inhibitory Boswellic Acid Analogues. J. Nat. Prod. 2000, 63 (8), 1058-1061. (5) Safayhi, H. Boswellic acids: novel, specific, nonredox inhibitors of lipoxygenase. J. Pharmacol. Exp. Ther. 1992, 261, 1143-1146. (6) Winterstein, S. Isolation from Olibanum tears. Z. Physiol. Chem. 1932, 208, 9. (7) Carey, M. C.; Small, D. M. Micellar properties of sodium fusidate, a steroid antibiotic structurally resemembling the bile salts. J. Lipid Res. 1971, 12, 604-613. (8) Sugihara, G.; Yamakawa, K.; Murata, Y.; Tanaka, M. J. Phys Chem. 1982, 86, 2748.
Figure 1. Structures of β-boswellic acid, acetyl β-boswellic acid, 11-keto-β-boswellic acid, and acetyl-11-keto-β boswellic acid.
Boswellic acids can be solublized and isolated by taking advantage of the phenomena of hydrotropy and that of the ability of hydrotropes to interact selectively with a solubilizate depending upon its structure. Hydrotropes, above a minimum concentration, form organized, albeit loose, microassemblies with large hydrophobic regions where the boswellic acids can be solubilized. The minimum hydrotrope concentration (MHC) is analogous to the critical micellar concentration (CMC) of a surfactant and is a characteristic of the hydrotrope. The dissolved solute can be recovered by diluting the aqueous extract solution below the MHC of the hydrotrope where the solute has negligible solubility. This simple recovery step along with little or no contamination of the product by the hydrotrope and potential reuse of the hydrotrope solution make the technique economically attractive for the extraction of thermally labile phytochemicals. Boswellic acids are sparingly soluble in water only to the extent of 17 mmol/ dm3, but their solubility in typical hydrotrope solutions can be increased severalfold.
10.1021/la034611r CCC: $25.00 © 2003 American Chemical Society Published on Web 07/31/2003
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Experimental Section Materials and Methods. n-Butyl benzene (obtained from Herdillia Chemicals Ltd., Mumbai) was sulfonated with concentrated sulfuric acid followed by neutralization with sodium hydroxide to prepare sodium n-butyl benzene sulfonate.9 Other hydrotropes, sodium cumene sulfonate and sodium p-toluene sulfonate, were obtained from Navdeep Chemicals Ltd., Mumbai, and were used as such. B. serrata gum resin was purchased locally. The resin was defatted with hexane and then solvent extracted with chloroform to determine the boswellic acids content of the resin. After removal of the solvent by evaporation, the residue was dissolved in methanol and acidified with 0.1 N hydrochloric acid to yield boswellic acids. The total boswellic acid content was found to be 76.5% (w/w) in the gum resin. A standard for boswellic acids was obtained from Hillgreen India Ltd. All other chemicals were of analytical reagent grade and were used without further purification. Solubility Studies. An excess amount of pure boswellic acids in powdered form was added to a 50 cm3 hydrotrope solution of known concentration in a stoppered conical flask. The suspension was stirred using a magnetic stirrer in a constant-temperature water bath (25 °C ( 1) for a period of 2 h to ensure the equilibrium. Samples were withdrawn from the solution at 15 min intervals and were diluted with water to bring the hydrotrope concentration below its MHC. During this dilution, the boswellic acids precipitated out as a white creamy nonsticky powder. The suspension was then centrifuged for 10 min at 2800g to separate the precipitated solid. The precipitate was air-dried and weighed. A small amount of the product was dissolved in chloroform for analysis on high-performance thin-layer chromatography (HPTLC). Extraction Studies. The extraction experiments were conducted in a fully baffled glass vessel (9.0 cm height and 7.0 cm i.d.) equipped with a 2.0 cm diameter six-bladed turbine impeller. The entire assembly was kept in a constant-temperature bath. Lumps of B. serrata resin were pulverized, and the particles of an average size of 50 µm were used for the extraction studies. A 10 gm sample was added to 100 cm3 of hydrotrope solution of known concentration, in the range of 50 mmol/dm3 to 2.0 mol/ dm3, in the glass vessel. The suspension was agitated vigorously at 1100 rpm for a period of 2 h. The solution was subsequently filtered using a vacuum. A clear pale brown solution was obtained as the filtrate, while the insoluble sticky solid portion of the oleoresin was collected as residue. The residue was washed with 10 cm3 of the fresh hydrotrope solution of the same concentration to remove any extract adhering to it. The filtrate was diluted with water to the respective MHC of the hydrotrope. A solid creamy white precipitate of total boswellic acids separated out, which was centrifuged at 2800g and finally air-dried to obtain a free-flowing cream-colored solid. This precipitate after dissolution in chloroform was analyzed by HPTLC. Molecular Modeling Studies. Hyperchem 7.0 (Hypercube Inc.) was used for simulation by molecular modeling of the hydrotropic solubilization of boswellic acids. The structures of the acids and hydrotrope were initially created and optimized individually for their intrinsic energy and other properties. The optimized structures were then used as starting points for subsequent calculations. After merging together the molecules of hydrotropes and the solute, the combined structure was optimized for the free energy using the AMBER94 force field. To solvate the solute, a Jorgensen’s Monte Carlo cubic box, 187.7 nm on a side, was used which contained 216 equilibrated water molecules, described by the TIP3P potential function.10 Analytical Procedure. The HPTLC analysis of the precipitate in chloroform was performed on 10 cm × 20 cm plates precoated with silica gel 60 F254 (Merck, FRG 5715). The samples were applied to the plates using Linomat IV according to the data pair technique. A chloroform-methanol (85:15% v/v) mixture was used as the eluent without chamber saturation. The chromatogram was developed to a distance of 9 cm and was then (9) Furniss, B. S.; Hannaford, A. J.; Rogers, V.; Smith, P. W. G.; Tatchell, A. R. Vogel’s Textbook of Practical Organic Chemistry, 4th ed.; Longman Scientific & Technical: Essex, U.K., 1978. (10) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. W. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 1983, 79 (2), 926-935.
Figure 2. Solubility of total boswellic acids in different hydrotrope concentrations (expected (s) and experimental (points)) (0, NaPTS; 4, NaCS; O, NaNBBS). sprayed with Libberman Burchard reagent.11 For quantification, calibration curves were prepared using standard boswellic acids as the external standard. The chromatograms were evaluated on the basis of peak areas measured at λ ) 630 nm using a CAMAG HPTLC unit equipped with a TLC Scanner-III densitometer operated with CATS software (version 3.04). After boswellic acids were precipitated out, the supernatant hydrotrope solution was filtered, acidified to pH 3.0, and then treated with the Libberman Burchard reagent to check complete precipitation of boswellic acids. The absence of color reaction indicated essentially complete precipitation of all triterpenoids from the hydrotrope solutions on dilution with water.
Results and Discussion Figure 2 shows the solubility of total boswellic acids in three hydrotrope solutions, sodium n-butyl benzene sulfonate (NaNBBS), sodium cumene sulfonate (NaCS), and sodium toluene sulfonate (NaPTS), in water. The alkyl benzene sulfonates have a definite and substantial effect on the solubility of the acids, which increased progressively with the hydrotrope concentration; the highest increase was seen with the most hydrophobic hydrotrope, NaNBBS. The aggregation of hydrotrope molecules has been recognized as a prerequisite for the increased solubility of an organic solute.12-16 If the solute is also capable of aggregating, then it may take part in the hydrotrope’s (11) Jayaraman, J. Laboratory Manual in Biochemistry, Biomolecules I: Carbohydrates; New Age International Ltd.: India, 1996; p 51. (12) 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, 2716. (13) Raman, G.; Gaikar, V. G. Extraction of piperine from Piper nigrum (black pepper) by hydrotropic solubilization. Ind. Eng. Chem. Res. 2002, 41, 2521-2528. (14) Srinivas, V.; Sundaram, C. S.; Balasubramanian, D. Molecular structure as a determinant of hydrotropic action: A study of polyhydroxybenzenes. Ind. J. Chem. 1991, 30B, 147-152. (15) Nagadome, S.; Okazaki, Y.; Lee, S.; Sasaki, Y.; Sugihara, G. Selective solubilization of Sterols by Bile Salt Micelles in Water: A thermodynamic study. Langmuir 2001, 17, 4405-4412. (16) Balasubramanian, D.; Srinivas, V.; Gaikar, V. G.; Sharma, M. M. Aggregation behaviour of hydrotropic compounds in aqueous solution. J. Phys. Chem. 1989, 93, 3865-3870.
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aggregation process. The formation of a more stable aggregate depends on its geometry, the presence of functional group(s), and the ability to coaggregate with hydrotrope molecules, which is influenced by the hydrophobic effect and charged interaction. An association model for hydrotrope solubilization considers stepwise aggregation of hydrotrope molecules forming n-mers.12
Hn-1 + H1 r f Hn
(1)
such that the aggregation constant (Kn) is defined as
Kn )
[Hn]
(2)
[Hn-1][H1]
Kn reduces with the increase in the aggregation number (n) and is related to the dimerization constant (K2) as
Kn ) K2/n
(3)
The total concentration of the hydrotrope can be related to hydrotrope monomer (H1) concentration in the solution through eqs 2 and 3.
Ct )
∑ iHi ) H1[2 exp(K2H1) - 1]
(4)
Each of the hydrotrope n-mers is capable of dissolving solute molecules by intercalation. A hydrotrope n-mer can also take up solute molecules in a stepwise manner, and the solute-hydrotrope aggregate association constant (Knm) is used to represent the association of a hydrotrope n-mer with m - 1 solute molecules and an additional solute molecule.
HnSm-1 + S r f HnSm
(5)
with Knm defined as
Knm )
[HnSm]
(6)
[HnSm-1][S]
It is further assumed that the successive addition of solute molecules decreases the association constant in the same manner as the hydrotrope’s own association behavior with the increase in the aggregation number, that is, Knm ) Ks/m where Ks characterizes the association of the first solute molecule with a solute-free hydrotrope n-mer which has been further assumed to be independent of the aggregation number of the n-mer. The total amount of solute (∆S) associated with hydrotrope aggregates of different aggregation numbers and each population of n-mer with the varying number of solute molecules associated with them from 1 to maximum n 1 can be obtained by the summation over all n-mers. n n-1
∆S )
HiSj ) 2(Ks/K2)[S1] {exp (K2H1) ∑2 ∑ j)1 (1 + K2H1)} (7)
where [S1] is the water solubility of the solute. A higher value of Ks signifies a larger hydrotropic effect. The Ks values for boswellic acid from the fit of the solubility data are 70.1, 64.2, and 18.7 dm3/mol for the three hydrotropes NaNBBS, NaCS, and NaPTS, respectively. The interaction of boswellic acid with NaNBBS seems to be stronger, followed closely by that with NaCS. The solubility curves, in both hydrotrope solutions, show almost a similar
behavior of boswellic acids. The hydrotrope’s self-association constant (K2) on the other hand is of a much lower magnitude (0.129 dm3/mol for NaNBBS, 0.126 dm3/mol for NaCS, and 0.122 dm3/mol for NaPTS). The hydrotrope self-association constant is 20% higher in the case of NaNBBS and NaCS and 50% higher for NaPTS as compared with the values obtained from piperine solubility studies in our earlier work.13 This is a clear indication of the effect of a solute on the aggregation tendency of a hydrotrope and of the fact that the solute plays a role in hydrotrope aggregation. Though the self-association of the hydrotrope is weaker, the hydrotrope’s aggregation tendency can increase in the presence of boswellic acids. The latter contributes to higher hydrophobicity within the coaggregates as compared to piperine which is a much smaller molecule. In the hydrotrope aggregate where boswellic acid is solubilized, the repulsion between the strongly ionic sulfonate headgroups is reduced, forming still tightly bound aggregates. The presence of somewhat loose aggregates in the case of piperine as compared to that with boswellic acid is also indicated by comparatively a much higher solubility of piperine in the hydrotrope solutions. Piperine solubility was enhanced by 230 times its solubility in water, while boswellic acid’s solubility was enhanced by only 120 times. The difference in the solubility values of boswellic acids in aqueous solutions of three alkyl benzene sulfonates can be attributed to the different sizes of their hydrophobic parts, the increase in the number of -CH2- groups in the hydrocarbon side chain, and the efficiency of intermolecular packing in their self-aggregates.14 Hydrotropic solubilization is a collective molecular phenomenon, and the self-aggregation of hydrotrope molecules in aqueous solutions is a prerequisite for the increased solubilization of organic compounds.16 The self-aggregation of hydrotrope molecules is a consequence of their amphiphilic nature, in a manner analogous to micelle-forming surfactants. It is, therefore, not surprising that the solubilization capacity of a hydrotrope is governed by its hydrophobic functionality, that is, the alkyl group on the aromatic sulfonates. This indicates that the hydrotropic solubilization is probably a consequence of the hydrophobic domains present within the hydrotrope aggregates, which provide a microenvironment compatible with the hydrophobic nature of boswellic acids. The association model explicitly considers the molecular aggregation. The association of a solute with the hydrotrope aggregates is reflected in the constant Ks, which determines the strength of interaction of the boswellic acids with different hydrotropes. The highest Ks is for the NaNBBS-boswellic acid aggregates, and hence the solubility of the boswellic acids is better in aqueous solution of the hydrotrope. The presence of boswellic acids may augment the hydrotrope association, as the process seems to be similar to molecular complexation. This indicates that the boswellic acids are very easily introduced into the aggregate structure without disrupting the aggregates; rather these molecules tend to stabilize the aggregates, the propensity of their final configuration leading to increased solubility. The solubilization of boswellic acids corresponds to the translation from the solid state to the solubilized state in the hydrotrope aggregates in aqueous solutions. We would like to consider the solubilization of boswellic acid per mole of hydrotrope (Sp), instead of the strict concept of solubilization by micelles. Based on the solubility concept, the Gibbs energy change is defined as15
∆G°s ) -RT ln (Sp)
(8)
Hydrotropic Solubilization of Boswellic Acids
Figure 3. Extraction of boswellic acids with different concentrations of NaNBBS (), 0.05 mol/dm3; 4, 0.5 mol/dm3; ×, 1.0 mol/dm3; O, 2.0 mol/dm3).
The term Sp is related to Ks between the hydrotrope aggregate and solute assuming that the aggregation number n is constant within a restricted hydrotrope concentration range. The Gibbs energy change can be used for relative comparison of the solubilization data in three hydrotrope solutions. ∆G°s is estimated to be -1.06 kJ/ mol for NaNBBS, -1.04 kJ/mol for NaCS, and -0.73 kJ/ mol for NaPTS. The negative Gibbs energy change shows translation from the bulk aqueous phase to the hydrotrope aggregate phase to be energetically favorable. The extraction of the boswellic acids from gum resin with different alkyl benzene sulfonates as hydrotropes was studied in the concentration range from 50 mmol/ dm3 to 2.0 mol/dm3. The extraction data were fitted in a rate equation of second order, and the fitted curves are shown for NaNBBS, NaCS, and NaPTS aqueous solutions at different concentrations (Figures 3-5). The sigmoidal nature of the curve indicates the extraction to be a secondorder process, that is, the rate is initially slow followed by a faster extraction stage before reaching a plateau. The final extraction limit was decided by the solubility of boswellic acids in the hydrotropic solutions under the given conditions. It appears that the extraction needs to overcome two resistances. The gum oleoresin of boswellia consists of a resinous amorphous mass. This gummy nature of the resin offers a resistance to penetration by the hydrotrope before enabling the solubilization of the boswellic acids into the hydrotrope aggregates. The rate of extraction should depend on how easily the hydrotrope can penetrate the resin matrix and enable the solubilization of boswellic acid. However, the initial lag period was smaller as boswellic acid was richly enmeshed in the solid gum resin, unlike in our earlier studies on the extraction of piperine from Piper nigrum, where a large amount of biomatrix had to be penetrated before the hydrotrope solutions could access piperine.13
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Figure 4. Extraction of boswellic acids with different concentrations of NaCS (), 0.05 mol/dm3; 4, 0.5 mol/dm3; ×, 1.0 mol/dm3; O, 2.0 mol/dm3).
Figure 5. Extraction of boswellic acids with different concentrations of NaPTS (), 0.05 mol/dm3; 4, 0.5 mol/dm3; 0, 1.0 mol/dm3; O, 2.0 mol/dm3).
The extraction efficiency of hydrotropes followed the order NaNBBS > NaCS > NaPTS in a given time of extraction. The efficiency of extraction is defined here as the ratio of the amount of the acids extracted into the hydrotrope solutions to that initially present in the raw material. A distinct relationship between the hydrophobic chain length of a hydrotrope and the selectivity of
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Figure 6. (a) Optimized structure of β-boswellic acid-NBBS after solvation, showing the conformation of the A, B, C, and D rings. (b) Optimized structure of β-amyrin-NBBS after solvation, showing the conformation of the A, B, C, and D rings.
extraction as indicated by the purity of the extracted acids was observed. The effective hydrophobic chain length varies from C5 for NaPTS to C8 for NaNBBS. The increased hydrophobicity provided by NaNBBS seems responsible for its higher extraction efficiency. The acids, because of their own large hydrophobic and smaller polar regions, intercalate between the hydrotrope molecules. Since the hydrophobicity of the aromatic sulfonates increases with the increase in alkyl group length, NaNBBS displays a greater efficiency for the solubilization of boswellic acids. From the hydrotrope-solute structures and interactions between them, it can be understood that the solute molecules intercalate between the hydrotrope molecules. The surface tension values of the hydrotrope solutions beyond their respective MHCs are 40 × 10-2 mN/m for NaNBBS, 43 × 10-2 mN/m for NaCS, and 53 × 10-2 mN/m for NaPTS.16 After the extraction of boswellic acids, the surface tensions of hydrotrope solutions decreased further to 32.6 × 10-2 mN/m for NaNBBS, 38 × 10-2 mN/m for NaCS, and 48 × 10-2 mN/m for NaPTS. The role of a solubilizate in lowering the interfacial tension of the hydrotrope solution can be visualized in the following way. The main limitation on the surface activity is the charge that is generated at the interface by adsorption of ionic
surface active molecules. However, since boswellic acid is uncharged its adsorption at the gas-liquid interface between the hydrotrope molecules is not impeded by the electric field. Its presence, instead, provides the further reduction of surface tension by reducing electrostatic repulsion between adjacent hydrotrope molecules. The better affinity of boswellic acids to the hydrotrope molecules results in exclusion of other triterpenes from the hydrotrope aggregates, increasing the selectivity of extraction. The selectivity of extraction was defined by the muchimproved purity of the boswellic acids in the extract. The purity of the acids obtained was the best from NaNBBS solutions (95%) followed by that obtained from NaCS solutions (93%). The sodium p-toluene sulfonate did not, however, show a similar selectivity toward the acids. It also showed poor capacity for solubilization of the boswellic acids. The impurities in the extract showed color formation with the Libberman Burchard reagent, suggesting that the impurities might be triterpenes such as β-amyrin and R-amyrin. To understand this interaction better and to explain the selective extraction of boswellic acids, different hydrotrope-boswellic acid combinations were simulated by
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Table 1. Heat of Formation of Hydrotropes, Solutes, and Hydrotrope-Solute Pairs hydrotrope/solutea NaNBBS β-BA β-AMY β-BA/NaNBBS β-AMY/NaNBBS a
heat of formation (kJ/mol)
change in heat of formation (kJ/mol)
-4.87 × 103 -5.47 × 103 -4.3 × 103 -1.06 × 104 -5.44 × 103
-2.6 × 102 3.7 × 103
BA ) boswellic acids; AMY ) amyrin.
molecular modeling calculations under solvated conditions.17 For the simulation, the molecular system was placed inside a box containing water molecules that imposes periodic boundary conditions allowing the molecules to move in a constant-density environment. The structurally optimized molecules were studied for their quantitative structure-activity relationship (QSAR) properties, viz., heat of formation, surface area, and molecular volume. In the case of the β-boswellic acid-hydrotrope (NBBS) pair, the heat of formation of the hydrotrope-solute pair was less than the sum of their individual heats of formation. The decrease for the β-boswellic acid-NBBS pair was greater than that for the β-amyrin-NBBS pair. A positive value in the latter case rather indicated its less stable nature and that the formation of such complexes was not thermodynamically feasible (Table 1). This can explain the selective extraction of boswellic acids as compared to that of β-amyrin. Even though structurally β-boswellic acids have the same basic β-amyrin triterpene framework, the polar area available with the β-boswellic acid molecule is 1.01 × 104 nm2 as compared to the polar area of the β-amyrin molecule, which is just 1.6 × 103 nm2. The overall stabilization of the hydrotrope aggregates is a combination of a favorable ionic network combined with hydrophobic layers clustering nonpolar regions. The cluster associations of the nonpolar organic regions form a hydrophobic layer that allows the association of remaining sulfonate groups with water. This involves oxygens from the sulfonate group as well as oxygen from water molecules leading to the extended hydrophilic network in two dimensions. Boswellic acids are easily solubilized in the hydrophobic layers of these microunits. The structure of boswellic acids has a hydroxyl group and a carboxylate group in solution, which can form additional H-bonds with the hydrophilic network associated with the hydrotrope aggregates. In effect, the boswellic acid molecules would be present in the hydrotrope aggregate with its hydrophobic region intercalated within the hydrophobic domain and the small polar region lying in the hydrophilic region of the aggregates. This affords an overall planar configuration similar to that of a liquid crystal. It is thus possible that in the solubilization process there is an interaction between the hydrophilic groups of the acid and the ionic region of the hydrotrope. The importance of polarity in the preferential solubilization can be explained using the molecular orbital calculations of the total dipole moments of the system.18 The net dipole moments for β-amyrin before and after solvation are 1.8 and 9.3 D, whereas the net dipole moments for β-boswellic acid, before and after solvation, are 4.6 and 10.2 D, respectively. The presence of the -COOH group on the boswellic acid molecule increases the net moment and (17) Maron, S. H.; Prutton, C. F. Principles of Physical Chemistry, India Ed.; MacMillan: New York, 1972; p 148-150. (18) Gale, M. M.; Saunders, L. The solubilization of steroids by lysophosphotidylcholine testosterine, estradiol and their 17 a-ethinyl derivative. Biochem. Biophys. Acta. 1971, 248, 466-470.
Figure 7. Electrostatic potential contours of (a) NBBS-BA, (b) CS-BA, and (c) PTS-BA.
thus enhances its interaction with hydrotrope assemblies. This is also reflected in the hydration energy values of β-boswellic acid (-27.5 kJ/mol) and β-amyrin (3.1 kJ/mol). The orientations and ring structures of the solubilizate affect the preferential solubilization characteristics.19 β-Boswellic acid showed a more penetrating trend into
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Table 2. Water-Accessible Surface Area of β-Boswellic Acid in Different Hydrotrope Solutions and Ks and K2 hydrotrope-solutea
Ks (dm3/mol)
K2 (dm3/mol)
WASA (nm2)
70.1 64.2 18.7
0.129 0.126 0.122
1.87 × 104 2.70 × 104 2.81 × 104
NaNBBS-BA Na-CS-BA Na-PTS-BA a
BA: β-boswellic acid.
the hydrophobic space within the aggregates. In the presence of n-butyl benzene sulfonate, the B, C, and D rings of β-boswellic acid adopt a chair conformation as compared to the boat conformation of the B, C, and D rings of β-amyrin [Figure 6a,b]. β-Boswellic acid has therefore a favorable positioning or orientation within the aggregates. Such an arrangement also explains the reduction in the surface tension of the hydrotrope solutions in the presence of boswellic acid. From the electrostatic potential surface (Figure 7a-c), it was seen that the β-boswellic acid molecule was completely trapped within the n-butyl benzene sulfonate aggregate, whereas the contribution of the cumene sulfonate aggregate was considerably less and with the p-toluene sulfonate aggregates it was the least. Since β-boswellic acid is completely enveloped within the NBBS aggregate, the higher solute-hydrotrope interaction constant (Ks) for β-boswellic acid-NBBS is justified, followed by those values with the β-boswellic acid-CS and β-boswellic acid-PTS pairs. This indicates that the hydrophobic surface area provided by n-butyl benzene sulfonate aggregates is larger than those in cumene sulfonate and p-toluene sulfonate aggregates. The water-accessible surface area (WASA) which is traced out by the probe sphere center as it rolls over the solute molecule, in this case the probe sphere being water, for the three hydrotropes also supports this conclusion (Table 2). The decrease in WASA is greater with the n-butyl benzene sulfonate aggregates followed by that with cumene sulfonate and p-toluene sulfonate aggregates, indicating that in aqueous solutions p-toluene sulfonate should form more open structures as compared to cumene sulfonate and n-butyl benzene sulfonate. The association models’ hydrotrope dimerization constant (K2) values are also in accordance. These indicate that even though the ionic headgroups cause repulsion, the boswellic acid molecule within the NBBS aggregates reduces the elec(19) Bhat, M.; Gaikar, V. G. Characterisation of interaction between Butyl Benzene Sulfonates and Cetyl Trimethylammonium Bromide in Mixed Aggregate systems. Langmuir 1999, 15, 4740-4751.
trostatic repulsion causing the hydrotrope molecules to interact more strongly. In the cumene sulfonate aggregates also, there was a significant contribution of the boswellic acid molecule to the tightening of the aggregate structure, but in the case of p-toluene sulfonate aggregates this contribution was observed to be relatively less. Hence, β-boswellic acid extraction shows the maximum selectivity during extraction using n-butyl benzene sulfonate solutions as compared with cumene sulfonate and p-toluene sulfonate solutions. Conclusions Boswellic acids, which are sparingly soluble in water, can be selectively extracted by using alkyl benzene sulfonate solutions. The efficiency of the extraction depends on the hydrophobic nature of the hydrotrope and also increases with its concentration. Since the water solubility of hydrotrope aggregates is significantly high, they provide a microenvironment of reduced polarity for the solubilizate. The layered structure of the hydrotrope aggregates provides easy access to the large nonpolar regions of boswellic acid molecules. The hydrophilic network bonds with the smaller polar region of the acid, which in turn stabilizes the aggregate layers, producing a cooperative solubilizing isotherm. Boswellic acids impart a high degree of stability to the host assembly, accounting for the unique selectivity aspect observed with hydrotropes. Acknowledgment. The authors acknowledge the support of this work from the Department of Science and Technology (Swarnajayanti Cell), G.O.I. Notations Ct ) hydrotrope concentration, mol/dm3 ∆G°s ) Gibbs free energy, kJ/mol Hf ) heat of formation, kJ/mol Hi ) concentration of hydrotrope i-mer, mol/dm3 K2 ) dimerization constant for hydrotrope, dm3/mol Ks ) solute-hydrotrope aggregate interaction constant, dm3/mol Kn ) hydrotrope aggregation constant (eq 2) Knm ) solute-hydrotrope aggregate association constant (eq 6) R ) gas constant, kJ/mol/K S1 ) water solubility of solute, mol/dm3 Sp ) solubility of boswellic acid per mole of hydrotrope ∆S ) increase in solubility in hydrotrope solutions, mol/ dm3 T ) temperature, K LA034611R
