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Ind. Eng. Chem. Res. 2009, 48, 4852–4857
PROCESS DESIGN AND CONTROL Effect of Extraction Solvent on the Bioactivity of an Herbal Formulation Kin N. Chan,† Choi C. Lau,† King L. Chow,‡ Kam M. Ko,§ Karl W. K. Tsim,‡ and Ka M. Ng*,† Departments of Chemical and Biomolecular Engineering, Biology, and Biochemistry, The Hong Kong UniVersity of Science and Technology, Clear Water Bay, Hong Kong
Zhikang Granules (ZKG) is a traditional Chinese medicine prescription for the treatment of hyperlipidemia symptoms. In this study, the effect of extraction solventssnamely, acetone, ethanol, and waterson the recovery of a bioactive ingredientsnamely, chrysophanolsand on the bioactivity of this herbal formulation was investigated. Although the solubility of chrysophanol is highest in acetone, the amount of chrysophanol extracted from Semen Cassiae is lowest in this solvent. Water was found to play an important role in the release of chrysophanol from Semen Cassiae. Two alternative ZKG processing methods are proposed based on the findings of bench-scale extraction tests. The resulting products were analyzed by both chemical analyses and biological assays. One of the products offered the same level of bioactivity while using only 10% of the original amount of Semen Cassiae. Introduction Herbal medicines and dietary supplements have been systematized, theorized, and used for health maintenance for thousands of years in China. Most traditional Chinese medicines (TCMs) are prepared by extracting the active ingredients from a concoction of herbs using primarily water and occasionally ethanol as the extraction solvent. The amount of an active ingredient, and thus the efficacy of the TCM, is expected to change if a different extraction solvent or different extraction conditions are used. Yet, relatively little has been done to explore this issue and to systematically optimize the extraction protocol to raise the yield. Because of the serious concern that unwanted compounds, instead of the targeted ingredients, could be extracted, resulting in a completely different or even harmful product, research in this direction has not been encouraged. This problem of self-inhibition is further compounded by the fact that the introduction of a new recipe often requires additional clinical trials that are time-consuming and expensive. Although it is prudent to strictly follow an established protocol, this is not very satisfying from a fundamental point of view. A better understanding of the relationship between extraction and bioactivity should lead to a more efficient manufacturing process and a better product in the long run. This study is an extension of that of Harjo et al. in which a systematic procedure was developed to synthesize process alternatives for manufacturing phytochemicals from herbs.1 The effect of extraction solvent on the bioactivity of a commercial TCM product, namely, Zhikang Granules (ZKG), was studied. First, the solubilities of the key active ingredient of ZKG, chrysophanol, in different solvents were measured. Then, benchscale extraction tests using the same set of solvents were performed to identify the best extraction conditions. The product quality was monitored by chemical analyses as well as the relevant in vitro and in/ex vivo bioassays. An improved * To whom correspondence should be addressed. Tel: 852 23587238. Fax: 852 23580054. E-mail:
[email protected]. † Department of Chemical and Biomolecular Engineering. ‡ Department of Biology. § Department of Biochemistry.
manufacturing process is proposed based on the new findings in the laboratory. Current Manufacturing Process ZKG consists of five herbs: Semen Cassiae, Fructus Lycii, Fructus Mori, Flos Carthami, and Fructus Crataegi. As demonstrated in clinical trials, ZKG is capable of lowering the total level of cholesterol and triglyceride in the plasma. Semen Cassiae is the principal herb of ZKG. Studies showed that a Semen Cassiae extract has plasma-lipid-level-modulating function in hyperlipidemia mice2,3 and could both reduce the weight and lower the serum triglyceride level in nutritive obesity rats.4,5 Hence, we focused on Semen Cassiae in this study. A two-stage extraction of Semen Cassiae is used in the current ZKG manufacturing process, as shown in Figure 1. Semen Cassiae is extracted in water at 95 °C for 20 min, and then, the mixture is kept at 25 °C overnight in the first extraction tank. Afterward, Semen Cassiae is separated out in a filtration tank. The first-stage liquid extract is kept for later use, and the solids are extracted again at 95 °C for 180 min in the second extraction tank. After filtration, as for the first-stage process, the secondstage liquid extract is stored for later use. Fructus Crataegi is extracted in 70% ethanol at 25 °C overnight. The ethanol content in the liquid extract is recovered in a solvent evaporator after filtration. The remaining herbs (Fructus Lycii, Fructus Mor, and Flos Carthami) are extracted together in boiling water for 180 min. All of these liquid extracts are grouped as the “other herbal extracts” and mixed with the first- and second-stage Semen Cassiae liquid extracts. Finally, the mixed solution is charged into a spray dryer in order to produce the final product in powder form. Solubility Measurements Chrysophanol, a bioactive anthraquinone of the principal herb Semen Cassiae,6 is the chemical marker for ZKG. Its solubilities in various solvents were measured using the isothermal soliddisappearance method7 (Figure 2). First, a known amount of chrysophanol with a purity of >98% (National Institute for the Control of Pharmaceutical and Biological Products, Beijing,
10.1021/ie8012538 CCC: $40.75 2009 American Chemical Society Published on Web 04/16/2009
Ind. Eng. Chem. Res., Vol. 48, No. 10, 2009
Figure 1. Schematic of the current ZKG manufacturing process.
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Figure 3. Solubilities of chrysophanol in various solvents.
Figure 4. HPLC chromatogram of Semen Cassiae liquid extract using 70% ethanol-water solution as the solvent.
Figure 2. Schematic diagram of the setup for SLE measurements.
China) and solvent were heated in a double-walled glass vessel to a temperature that was 5 °C higher than the target temperature. After 10 min of equilibration, a known amount of solvent was added to the glass vessel slowly until the solid chrysophanol completely dissolved and the mixture became a homogeneous solution. Afterward, the homogeneous solution was cooled until the solute was observed to crystallize from the solution. The temperature was then raised to the target temperature and kept there for 10 min. Next, a small but known amount of solvent was added to the system again every 5 min until all of the solute was dissolved. The solubility of chrysophanol at the target temperature was calculated from the amount of solute and the total amount of solvent in the glass vessel. The solubilities of chrysophanol in acetone, an aqueous solution of 70 vol% ethanol, and methanol were determined for temperatures ranging from 20 to 50 °C (Figure 3). Among these three organic solvents, the relative solubility of chrysophanol was found to be the highest in acetone, followed by methanol and then 70% ethanol. Also measured was the solubility of chrysophanol at 95 °C in water, the sole solvent in the current Semen Cassiae extraction process. The solubility value of 0.03 mg of chrysophanol/g of water was much lower than those observed in the organic solvents. Extraction of Chrysophanol from Semen Cassiae A series of bench-scale extraction tests were conducted using acetone, 70% ethanol, and water as extraction solvents. Methanol was not used in these experiments because it has a lower chrysophanol solubility than acetone. Also, according to the ICH
(International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use) guidelines on the amount of acceptable intake of residual solvents in pharmaceuticals for the safety of the patient, whereas ethanol and acetone are class 3 solvents with low toxic potential, methanol is a class 2 solvent that is suspected of some significant but reversible toxicities.8 In these extraction experiments, all herbs were supplied by Tianda Pharmaceuticals Co. Ltd. After being dried in a vacuum oven at 50 °C overnight, they were ground into a powder with a particle size of
