A Nanomized Nutraceutical with Enhanced Bioactivity and Bioavailability

May 9, 2012 - such as particle size and appearance were studied using orange oil and Oil Red O as model compounds. The relationship between...
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Product Design: A Nanomized Nutraceutical with Enhanced Bioactivity and Bioavailability Yeuk Tin Lau,† Na Chen,‡ Kam Ming Ko,‡ and Ka Ming Ng*,§ †

Bioengineering Graduate Program, ‡Section of Biochemistry and Cell Biology, Division of Life Science, and §Department of Chemical and Biomolecular Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong ABSTRACT: This study considered the design and process development of nanomized products in two product forms: nanoemulsion and nanodispersible solids. The effect of product formulation and processing conditions on product attributes such as particle size and appearance were studied using orange oil and Oil Red O as model compounds. The relationship between particle size and product quality in terms of bioactivity and bioavailability was illustrated using schisandrin B, a strong antioxidant, as a model drug. The bioactivity as measured by in vivo glutathione regeneration capacity assay of schisandrin B with an average size of 45 nm was nearly double that of the original coarse schisandrin B; the corresponding bioavailability as measured by the plasma schisandrin B level in an animal study was 14.5-fold higher.

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INTRODUCTION About 40% of the drugs from natural sources and 60% of the chemically synthesized drugs are poorly water-soluble.1 Most of these drugs show poor bioavailability, especially those administered through the oral route.2 However, a wide variety of techniques ranging from emulsification to high-pressure homogenization can be used to nanomize these drugs;3−5 examples include DissoCubes6 and Iota NanoSolutions.7 Nanomized drugs such as danazol,8 amphotericin B,9 and curcumin10 have been shown to offer enhanced bioavailability and sometimes bioactivity. To reduce the time-to-market, it is highly desirable to design such a nanomized product in a systematic way. Recently, a large number of research papers have considered various aspects of product design and development.11−16 A systematic methodology involving computer-aided design, experimental planning, and testing has been developed for formulated products.17,18 Experimental testing in product design is also emphasized in the integrative approach proposed by Cheng et al.;19,20 two products, a skin care cream and a transdermal patch containing a traditional Chinese medicinal tincture, were developed to illustrate their approach. The objective of this study is to extend this integrative approach to the design and process development of two forms of a nanomized product: nanoemulsion and nanodispersible solids. Three poorly water-soluble substances, including orange oil, Oil Red O, and schisandrin B (Sch B), served as model compounds in this study. Orange oil and Oil Red O are readily available; they are used to study the impact of materials selection and operating conditions on product attributes and stability. Sch B is bioactive but requires significant effort to extract and purify. Only Sch B is used for studying the enhancement in bioavailability and bioactivity due to nanomization. The generalized framework discussed in this paper can be used to produce other nanoemulsions and nanodispersible solids. Process for the Formation of Nanoemulsion and Nanodispersible Solids. The nanomization process works as © 2012 American Chemical Society

follows. An organic solution was prepared by dissolving the poorly water-soluble material in an organic solvent under gentle stirring for 10 min. An aqueous solution was similarly prepared by dissolving a surfactant under gentle stirring for 10 min. After that, the two solutions were mixed together and its pH was adjusted to a desired value using nitric acid (1.0 M) or sodium hydroxide (2.0 M). Homogenization was first performed using a hand-held homogenizer (T25 digital, IKA Ultra-Turrax) followed by a benchtop high-pressure homogenizer (Panda 2K, NS 1001 L, GEA NiroSoavi). For the former, 30 mL of the aqueous− organic mixture was homogenized for a specified period of time at 20−40 °C. For the latter, 300 mL of the emulsion from the hand-held homogenizer was subjected to high-pressure homogenization at 200−1120 bar and 20−40 °C for a number of cycles to obtain the nanoemulsion. Nanodispersible solids of poorly water-soluble material were produced from the nanoemulsion. The recipe was the same as that described above except that a polymeric agent was also included in the aqueous solution. The nanoemulsion preparation was then sprayed into an aluminum container immersed in a liquid nitrogen bath, using a setup shown in Figure 1. All frozen particles of the nanoemulsion were collected. Next, the frozen particles were placed in a freezedryer overnight and lyophilized to obtain nanodispersible solids in dry powder form.

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MATERIALS Sch B was purified from a petroleum ether extract of Fructus Schisandrae (FS).21 Orange oil (Kosher grade, extracted from Citrus sinensis), limonene (97% purity), polyethylene glycol (PEG, average molecular weight 3 350), polyvinylpyrrolidone (PVP, average molecular weight 10 000), and tert-butyl Received: Revised: Accepted: Published: 7320

August 23, 2011 April 16, 2012 May 9, 2012 May 9, 2012 dx.doi.org/10.1021/ie201886p | Ind. Eng. Chem. Res. 2012, 51, 7320−7326

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Figure 1. Experimental setup of the spray cooling process.

Figure 2. LC/MS/MS chromatogram of 0.5 mg/L Sch B.

hydroperoxide (tBHP) were purchased from Sigma-Aldrich. Polysorbate 80 (PhEur grade) and Oil Red O were purchased from Fluka. Lactose was purchased from Farco Chemical Supplies (China). Except for the chemicals otherwise mentioned, all chemicals were of analytical grade. Product Characterization by Particle Size and Composition. The mean particle size of dispersed particles based on number was determined using laser light scattering correlation spectroscopy (90 Plus/BI-MAS, Brookhaven Instruments Corporation). The concentration of a compound in the nanoemulsion or the nanodispersible solids was determined using a Waters Alliance HPLC system which was comprised of the Waters 2956 separation module and the Waters 2996 photodiode array detector. For Sch B, a Nova-Pak C18 4 μm (3.9 mm × 300 mm) column was used. The mobile phase was acetonitrile and water (55:45, v/v) maintained at a flow rate of 1 mL/min. A 20 μL sample was injected, and the elution was monitored by UV absorbance at 254 nm. For limonene, an Agilent Zorbax Extend-C18 5 μm (4.6 mm × 250 mm) column was used. The mobile phase was acetonitrile and water maintained at a flow rate of 0.8 mL/min with a gradient elution. A 20 μL sample was injected, and the elution was monitored by UV absorbance at 214 nm. The amount of Sch B in the blood plasma of rat was determined using liquid chromatography/mass spectrometry/ mass spectrometry (LC/MS/MS). The analysis was carried out using an Agilent 6410 triple quadruple mass spectrometer equipped with a series 1290 HPLC system consisting of a quaternary pump, automatic solvent degasser, auto sampler, and an automatic thermostatic column compartment. An

Figure 3. (a) Mean particle size of nanoemulsion at different time durations of mechanical homogenization. (b) Mean particle size of nanoemulsion at different number of homogenization cycles under 1000 bar pressure in high-pressure homogenization. (c) Mean particle size of nanoemulsion at 6, 12, and 18 homogenization cycles under various pressures in high-pressure homogenization.

Figure 4. Mean particle size of nanoemulsion at different S/O ratios.

Agilent Zorbax Extend-C18 5 μm (4.6 mm × 250 mm) with a 0.5 online filter (Upchurch Scientific Ltd.) was used. The mobile phase, methanol/water containing 0.1% formic acid (70:30, v/v), was run at a flow rate of 0.5 mL/min with an operating temperature of 25 °C. Positive ion electrospray ionization (ESI) was used to form sodium adduct molecular ions in selected reaction monitoring mode at m/z 401.2−331.1 7321

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Figure 5. Mean particle size of nanoemulsion at different pH.

Table 1. Mean Particle Size of Nanoemulsions and Nanodispersible Solids of Orange Oil, Oil Red O, and Sch B chemical orange oil Oil Red O

Sch B

polymeric agent

mean particle size of nanoemulsion (nm)

mean particle size of nanodispersible solids (nm)

PVP lactose PEG PVP PVP

32.2 21.6 33.2 27.1 32.3

14.5 26.5 20.2 21.6 29.7

and 401.2−300.1 for Sch B. The optimum ESI conditions for these compounds included a nitrogen nebulizer pressure of 35 psi, a nitrogen-drying gas temperature of 325 °C at 10 L/min, spray voltage of 4000 V. A LC/MS/MS chromatogram of 0.5 mg/L Sch B in methanol is shown in Figure 2. Product Quality by Biological Assays. Adult female Sprague−Dawley rats (8−10 weeks old; 200−250 g) were maintained under a 12 h dark/light cycle in an air/humidity controlled room at about 22 °C and allowed water and food ad libitum. All experimental protocols were approved by The Committee for Research Practice, The Hong Kong University of Science & Technology. For bioavailability tests, animals were randomly divided into groups of 4 animals. A single oral dose of coarse, 45 nm, or 168 nm nanomized Sch B at a dose of 34.4 mg/kg was administered to these animals. Blood samples were collected from rats at increasing time intervals of 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, and 36 h after the oral dosing. Blood samples of about 0.25 mL were collected in heparinized 1.5 mL polythene tubes by tail bleeding. After centrifuging the whole blood at 400g for 15 min, 0.1 mL of plasma sample was collected and stored at −80 °C until analysis. For bioactivity tests, rats were intragastrically administered with coarse or nanomized Sch B. A time of 24 h after the last dosing, heart ventricular and liver tissues were obtained from pentobarbital-anesthetized animals. Next, heart ventricular and liver tissue samples were rinsed with ice-cold homogenizing buffer (50 mM Tris 0.1 mM EDTA, pH 7.6). A 10% (w/v) cardiac and hepatic homogenates were prepared by homogenizing the respective tissue sample with Polytron homogenizer at 135 000 rpm for two 10 s bursts on ice. Then, aliquots (2 mL) of tissue homogenates were mixed with an equal volume of tBHP solution. Increasing concentrations of tBHP were used for cardiac homogenates (0.025 and 0.050 mM final concentration in isotonic saline) and hepatic homogenates (0.1 and 0.2 mM). The reaction mixtures were then incubated for increasing periods of time (15 s to 60 min) at 37 °C. At the

Figure 6. Nanodispersible solids of Oil Red O formed by using (a) lactose, (b) polyethylene glycol, and (c) polyvinyl pyrrolidone.

end of each time point, the reaction was terminated by mixing a 0.4 mL aliquot of the reaction mixture with 0.1 mL of cold trichloroacetic acid (TCA) (25%, w/v), and the reaction mixtures were then centrifuged at 2500g for 10 min at 4 °C. The basal thiol level, an indirect measure of reduced glutathione (GSH), of cardiac or hepatic homogenate was measured using the supernatant prepared from a mixture containing 0.2 mL of the respective tissue homogenate, 0.2 mL of saline, and 0.1 mL of TCA solution. An aliquot (100 μL) of supernatant was added into 0.5 mL of sodium phosphate buffer (0.1 M, pH 8.0), and the mixture was mixed by vortexing. An aliquot of 25 μL of freshly prepared 5,5′-dithionitrobenzene (3 mM) was added to the reaction mixture followed by thorough mixing. Then all reaction mixtures were incubated at 37 °C for 7322

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Table 2. Optimized Nanomization Composition of Orange Oil, Oil Red O, and Sch B Formulations orange oil component

volume (mL)

Polysorbate 80 orange oil PVP water

31.50 21.98 N/A 146.52

mass (g)

33.52 18.53 29.30 146.52 Oil Red O

concentration (wt %) 14.71 8.13 12.86 64.30

component

volume (mL)

mass (g)

concentration (wt %)

Polysorbate 80 limonene PVP water Oil Red O

38.71 19.35 N/A 141.94 N/A

41.19 16.32 31.86 141.94 0.03

17.80 7.05 13.77 61.36 0.01

component

volume (mL)

mass (g)

concentration (wt %)

Polysorbate 80 limonene PVP water Sch B

38.16 21.11 N/A 140.73 N/A

40.60 17.80 27.73 140.73 2.86

17.68 7.75 12.07 61.26 1.24

Sch B

Figure 8. (a) Stability of nanoemulsion at different pH. (b) Stability of nanodispersible solid at different pH.

Figure 7. Stability of nanodispersible solid in aqueous redispersion.

10 min. The absorbance at 412 nm of the reaction mixture was measured. Glutathione regeneration capacity (GRC) was estimated by computing the area under the curve (AUC) of the graph plotting the percentage of basal GSH level against the incubation time (up to 60 min). Impact of Operating Conditions and Selection of Materials. Effect of Homogenizer Operating Conditions. For mechanical homogenization, the particle size of the nanoemulsion depends on the time duration of homogenization (tHomo) and the rotational speed (ω). Figure 3a shows the dependence of the mean particle size of the orange oil at different time durations of mechanical homogenization at a rotational speed of 24 000 rpm. For high-pressure homogenization, the particle size depends on the number of homogenization cycles (n) and applied pressure (ΔpHomo); Figure 3b shows the results at 1000 bar of applied pressure. It was observed that the two homogenization processes showed a similar trend of change. At the beginning, the mean particle size decreased with an increase in time duration or number of the homogenization cycle and reached the minimum particle size of around 30 nm after 30 min of mechanical homogenization or after 15 cycles of high-pressure homogenization. Beyond that, the particle size increased slightly with further homogenization. In general, the mean particle size of the nanoemulsion is

Figure 9. Plasma concentration versus time curves of 45 nm, 168 nm, and coarse Sch B after oral administration at a dose of 34.4 mg Sch B/ kg rat. Each point represents the mean ± SD of 4 rats.

Table 3. Pharmacokinetic Parameters of 45 nm, 168 nm, and Coarse Sch B after Oral Administration at a Dose of 34.4 mg Sch B/kg Rat parameters

45 nm Sch B

168 nm Sch B

coarse Sch B

AUC (mg/L h) Cmax (mg/L) tmax (h)

19.76 1.44 4

1.53 0.09 4

1.36 0.21 3

determined by the balance between two counteracting factors, droplet break-up, and recoalescence.22−24 The trend reversal beyond a particle size of around 30 nm simply implied that recoalescence dominated. Figure 3c shows the dependence of the mean particle size on applied pressure in the range of 6−18 homogenization cycles. It can be seen that the mean particle size of the sample under higher applied pressures was smaller than those under lower applied pressures. This observation agreed well with the suggestions of Floury et al.22and Schultz et 7323

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Figure 10. Enhancement of glutathione regeneration capacity (GRC) by different types of Sch B in (a) cardiac and (b) hepatic tissues at a dose of 10 mg/kg/day for 14 days.

Figure 12. Size dependence of glutathione regeneration capacity (GRC) enhancement by nanomized Sch B in (a) cardiac and (b) hepatic tissues.

al.25 that the efficiency of oil droplet disruption inside the homogenizer increases with the applied pressure. Effect of Surfactant-to-Oil (S/O) Ratio. The nanomization process is highly dependent on the amount of surfactant. Figure 4 shows the effect of S/O ratio on the mean particle size of orange oil with the mechanical homogenizer operating at 24 000 rpm for 20 min. The particle size dropped from 270 nm to the minimum of 30 nm, when the S/O ratio increased from 0.6 to 5. In this range, a kinetically stable nanoemulsion system was formed. The particle size increased from 30 to 900 nm when the S/O ratio was further increased from 5 to 7.2. In this range, the additional surfactant molecules formed aggregates on their own. S/O ratios beyond 7.2 had also been investigated, but no nanoemulsion was formed. Effect of System pH Value. Figure 5 shows the mean particle size of the nanoemulsion formed by mechanical homogenization under different pH values. The mean particle size was close to 20 nm at low pH and around 370 nm at high pH. The surfactant used, polysorbate 80, has 20 ether groups. At low pH, the protonation of oxygen atoms in the ether functional groups would increase both the hydrophilicity and intermolecular repulsion of the surfactant molecules. This change favored the formation of small oil droplets during nanomization. Insufficient protonation at high pH caused a significant increase in mean particle size of oil droplets.

Figure 11. Enhancement of glutathione regeneration capacity (GRC) by nanomized 45 nm and coarse Sch B in (a) cardiac and (b) hepatic tissues at different doses for 3 days. 7324

dx.doi.org/10.1021/ie201886p | Ind. Eng. Chem. Res. 2012, 51, 7320−7326

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Selection of Polymeric Agent. For the formation of a nanodispersible solid, a water-soluble and edible polymeric agent is used in the product formulation to provide a matrix for the solid form and prevent the aggregation of the nanosized particles. Lactose, polyethylene glycol (PEG), and polyvinyl pyrrolidone (PVP) were considered in this study. A number of trials were performed to fix the product formulation. The operating conditions were fixed based on the results reported above as follows: ΔpHomo = 1000 bar, n = 12 cycles, S/O ratio = 2.2−2.5, and pH = 5. The mean particle sizes of nanoemulsion and redispersed particles from the nanodispersible solids are reported in Table 1. It should be noted that the mean particle size of orange oil solid product (14.5 nm) was nearly half of its size in the nanoemulsion (32.2 nm). This may be due to the fact that the volatile components in orange oil were partly removed during the freeze-drying process. This was confirmed by an experiment which showed that the amount of orange oil decreased after freeze-drying. As can be seen with the data for Oil Red O, all three polymeric agents could prevent aggregation of particles during storage. The products with lactose and PEG were agglomerated and bulky whereas the product with PVP was loose and fine (Figure 6). Thus, PVP was chosen as the polymeric agent in formulation design because of the superior physical appearance of the product, which is an important product attribute. The concentration of polymeric agent affects both the ability to resist aggregation and the final product appearance. After additional iterations, the final formulations with around 13 wt % PVP are given in Table 2. Stability of Nanomization Products. All three example products exhibited good stability during storage. It was observed that the mean particle size of the nanoemulsion of orange oil (produced by high-pressure homogenization under 1120 bar applied pressure) was stable even after 8 months of storage at room temperature. Furthermore, nanodispersible solids also showed good stability during storage at room temperature. The nanodispersible solids of Sch B, stored in a glass container sealed with parafilm at room temperature after production, were redispersed at different times during a 3month storage in water. The particle size was found to be the same as that at the beginning of storage. Figure 7 shows that the particle size remained constant within the first 24 h but progressively increased after 24 h. This degree of stability was sufficiently long for orally administered drugs. For oral administration, the nanomization product should also exhibit good stability in an acidic environment. Figure 8 shows that the orange oil nanoemulsion was stable in the pH range from 2 to 10 while Sch B was stable from 2 to 7, beyond which there was a rapid increase in particle size in both nanomization products. Bioavailability Enhancement of Nanomized Sch B. Sch B is the most abundant, active dibenzocyclooctadiene derivative isolated from FS. Sch B treatment was found to protect against oxidant-induced injury in rodent liver and heart.26 Both the cardio- and hepatoprotection were likely mediated by the enhancement of mitochondrial glutathione status.27 According to Ko and co-workers,28 Sch B is able to enhance the GRC, an indirect measure of glutathione antioxidant status, of cardiac and hepatic tissue homogenates. However, because of its low aqueous solubility, Sch B faces a formulation challenge for oral administration. As such, Sch B as well as the formulation reported in Table 2 is a suitable model compound to demonstrate the product design issues related to nanomization and product formulation.

Figure 9 shows the time-dependent changes in mean plasma concentration of Sch B after the oral administration of 45 nm Sch B, 168 nm Sch B, and coarse Sch B. The pharmacokinetic parameters in Figure 9, AUC, Cmax (maximum Sch B concentration), and tmax (time at which Cmax occurs), are summarized in Table 3. Relative bioavailability describes the fraction of an administrated dose of drug that is absorbed and undergoes the systemic circulation. Under an equal dosage condition, the relatively bioavailability of the nanomized Sch B product with respect to the coarse Sch B, FNano/Coarse, can be calculated as follows: FNano/Coarse =

AUC Nano AUCCoarse

(1)

where AUCNano and AUCCoarse are the areas under the curve of the plasma concentration versus time curves for the nanomized Sch B and coarse Sch B, respectively. The relatively bioavailability of 45 nm Sch B and 168 nm Sch B were 14.5 and 1.1, respectively. The results clearly indicated that nanomization could significantly improve the bioavailability of Sch B. Bioactivity Enhancement of Nanomized Sch B. tBHP is commonly used for investigating the pharmacological mechanism involved in oxidative tissue damage. When it is exposed to tissues, tBHP is decomposed by the catalytic action of glutathione transferases, with resultant formation of tert-butyl alcohol and concomitant oxidation of GSH to oxidized glutathione (GSSG).29,30 The area under the curve of a graph plotting the percent GSH level (with respect to the basal value) in the tissue homogenate against time (min) post-tBHP challenge was computed and viewed as GRC, which is the measurement of the bioactivity of Sch B. Figure 10 shows the changes in GRC of cardiac and hepatic tissue homogenates prepared from animals pretreated with nanomized 45 nm Sch B and coarse Sch B (10 mg/kg/day × 14 days). In cardiac tissues, the extent of stimulation on GRC produced by nanomized 45 nm Sch B (24% above control) was larger than that of coarse Sch B (13% above control). A similar trend was observed in hepatic tissues, with the extent of GRC stimulation being 23% and 10% above the control for the nanomized 45 nm and coarse Sch B, respectively. The results confirmed nanomization improved the bioactivity of Sch B in vivo. To investigate the effect of the amount of Sch B, animals were treated with larger daily doses (17.2 and 34.4 mg/kg) of nanomized 45 nm Sch B for 3 days. As shown in Figure 11, the extent of stimulation on GRC by nanomized Sch B was about double that of the coarse Sch B in both cardiac and hepatic tissues at all tested doses. The dependence of bioactivity enhancement, in terms of GRC enhancement, on particle size was also investigated. As shown in Figure 12, the bioactivity enhancement of the 168 nm Sch B preparation was smaller than that of the 45 nm Sch B preparation. This size dependence may be attributed to the differential intestinal absorption rate of the Sch B in the gastrointestinal tract. Following the intragastric administration, Sch B is transferred to the small intestine where the absorption into the bloodstream takes place. Since a smaller Sch B particle has a higher surface area to mass ratio, it has a higher rate of transport in the absorptive direction. 7325

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(12) Whitnack, C.; Heller, A.; Frow, M. T.; Kerr, S.; Bagajewicz, M. J. Financial risk management in the design of products under uncertainty. Comput. Chem. Eng. 2009, 33, 1056. (13) Street, C.; Woody, J.; Ardila, J.; Bagajewicz, M. Product design: A case study of slow-release carpet deodorizers/disinfectants. Ind. Eng. Chem. Res. 2008, 47, 1192. (14) Marquardt, W.; Harwardt, A.; Hechinger, M.; Kraemer, K.; Viell, J.; Voll, A. The biorenewables opportunitytoward next generation process and product systems. AIChE J. 2010, 56, 2228. (15) Hechinger, M.; Voll, A.; Marquardt, W. Towards an integrated design of biofuels and their production pathways. Comput. Chem. Eng. 2010, 34, 1909. (16) Fung, K. Y.; Kwong, C. K.; Siu, K. W. M.; Yu, K. M. A multiobjective genetic algorithm approach to rule mining for affective product design. Expert Syst. Appl. 2012, 39, 7411. (17) Conte, E.; Gani, R.; Ng, K. M. Design of formulated products: A systematic methodology. AIChE J. 2011, 57, 2431. (18) Conte, E.; Gani, R.; Cheng, Y. S.; Ng, K. M. Design of formulated products: Experimental component. AIChE J. 2012, 58, 173. (19) Cheng, Y. S.; Lam, K. W.; Ng, K. M.; Ko, K. M.; Wibowo, C. An integrative approach for product developmenta skin-care cream. Comput. Chem. Eng. 2009, 33, 1097. (20) Cheng, Y. S.; Ng, K. M.; Wibowo, C. Product design: A transdermal patch containing a TCM tincture. Ind. Eng. Chem. Res. 2010, 49, 4904. (21) Ip, S. P.; Poon, M. K. T.; Wu, S. S.; Che, C. T.; Ng, K. H.; Kong, Y. C.; Ko, K. M. Effect of schisandrin B on hepatic glutathione antioxidant system in mice: Protection against carbon tetrachloride toxicity. Planta Med. 1995, 61, 398. (22) Floury, J.; Legrand, J.; Desrumaux, A. Analysis of a new type of high pressure homogenizer. Part B. study of droplet break-up and recoalescence phenomena. Chem. Eng. Sci. 2004, 59, 1285. (23) Perrier-Cornet, J. M.; Marie, P.; Gervais, P. Comparison of emulsification efficiency of protein-stabilized oil-in-water emulsions using jet, high pressure and colloid mill homogenization. J. Food Eng. 2005, 66, 211. (24) Tesch, S.; Schubert, H. Influence of increasing viscosity of the aqueous phase on the short-term stability of protein stabilized emulsions. J. Food Eng. 2002, 52, 305. (25) Schultz, S.; Wagner, G.; Urban, K.; Ulrich, J. High-pressure homogenization as a process for emulsion formation. Chem. Eng. Technol. 2004, 27, 361. (26) Chiu, P. Y.; Ko, K. M. Time-dependent enhancement in mitochondrial glutathione status and ATP generation capacity by schisandrin B treatment decreases the susceptibility of rat hearts to ischemia-reperfusion injury. BioFactors 2003, 19, 43. (27) Yim, T. K.; Ko, K. M. Schisandrin B protects against myocardial ischemia-reperfusion injury by enhancing myocardial glutathione antioxidant status. Mol. Cell. Biochem. 1999, 196, 151. (28) Ko, K. M.; Mak, H. F.; Li, P. C.; Poon, K. T.; Ip, S. P. Enhancement of hepatic glutathione regeneration capacity by a lignanenriched extract of fructus schisandrae in rats. Jpn. J. Pharmacol. 1995, 69, 439. (29) Sies, H.; Summer, K. H. Hydroperoxide-metabolizing systems in rat liver. Eur. J. Biochem. 1975, 57, 503. (30) Sies, H.; Gersterecker, C.; Menzel, H.; Flohe, L. Oxidation in the NADPH system and release of GSSG from hemoglobin free perfused rat liver during peroxidatic oxidation of glutathione by hydroperoxide. FEBS Lett. 1972, 27, 171. (31) Harjo, B.; Wibowo, C.; Zhang, E. J. N.; Luo, K. Q.; Ng, K. M. Development of process alternatives for separation and purification of isoflavones. Ind. Eng. Chem. Res. 2007, 46, 181. (32) Zhang, E. J. N.; Ng, K. M.; Luo, K. Q. Extraction and purification of isoflavones from soybeans and characterization of their estrogenic activities. J. Agric. Food Chem. 2007, 55, 6940.

CONCLUSIONS An integrative approach for product design and development19 has been applied to the formulation, stability testing, and performance testing of nanomized products. In product formulation, it was shown that the duration of homogenization, operating pressure, S/O ratio, and pH had considerable impact on the final product. In materials selection, PVP offered the best appearance and was the material of choice for the nanodispersible solids. The nanomization products were stable during storage and in an acidic environment. In addition to serving as a case study on product design, this investigation showed for the first time the enhancement of Sch B bioavailability and bioactivity by nanomization. These findings would form the basis for extending this study to consider the commercialization of this product as well as other nutraceuticals.31,32

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AUTHOR INFORMATION

Corresponding Author

*Phone: 852 23587238. Fax: 852 23580054. E-mail: kekmng@ ust.hk. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Research Grant Council (Grant 605208) is gratefully acknowledged. We thank Christianto Wibowo of CWB Technology for many suggestions throughout the course of this work and Dr. Yue Zhu for his help on LC/ MS/MS analysis.

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REFERENCES

(1) Lipinski, C. A. Poor aqueous solubilityan industry wide problem in drug discovery. Am. Pharm. Rev. 2002, 5, 82. (2) Merisko-Liversidge, E.; Liversidge, G. G.; Cooper, E. R. Nanosizing: A formulation approach for poorly-water-soluble compounds. Eur. J. Pharm. Sci. 2003, 18, 113. (3) Keck, C. M.; Müller, R. H. Drug nanocrystals of poorly soluble drugs produced by high pressure homogenization. Eur. J. Pharm. Sci. 2006, 62, 3. (4) Kesisoglou, F.; Panmai, S.; Wu, Y. Nanosizingoral formulation development and biopharmaceutical evaluation. Adv. Drug Delivery Rev. 2007, 59, 631. (5) Gutiérrez, J. M.; González, C.; Maestro, A.; Solè, I.; Pey, C. M.; Nolla, J. Nano-emulsions: New applications and optimization of their preparation. Curr. Opin. Colloid Interface Sci. 2008, 13, 245. (6) Müller, R. H.; Becker, R.; Kruss, B.; Peters, K. Pharmaceutical nanosuspensions for medicament administration as systems with increased saturation solubility and rate of solution. U.S. Patent 5,858,410, January 12, 1999. (7) Cooper, A.; Butler, R. Porous material and method of production thereof. International Patent WO 2005/014,704 A1, 2005. (8) Liversidge, G. G.; Cundy, K. C. Particle size reduction for improvement of oral bioavailability of hydrophobic drugs: I. Absolute oral bioavailability of nanocrystalline danazol in beagle dogs. Int. J. Pharm. 1995, 125, 91. (9) Kayser, O.; Olbrich, C.; Yardley, V.; Kiderlen, A. F.; Croft, S. L. Formulation of amphotericin B as nanosuspension for oral administration. Int. J. Pharm. 2003, 254, 73. (10) Bisht, S.; Feldmann, G.; Soni, S.; Ravi, R.; Karikar, C.; Maitra, A. Polymeric nanoparticle-encapsulated curcumin (“nanocurcumin”): A novel strategy for human cancer therapy. J. Nanobiotechnol. 2007, 5, 3. (11) Voncken, R. M.; Broekhuis, A. A.; Heeres, H. J.; Jonker, G. H. The many facets of product technology. Chem. Eng. Res. Des. 2004, 82, 1411. 7326

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