Separation and Purification of Schisandrin B from Fructus Schisandrae

May 15, 2008 - and fatty oils.4 Table 1 summarizes the major classes of chemicals and their relative composition in FS. Dibenzocyclooctadiene lignans,...
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Ind. Eng. Chem. Res. 2008, 47, 4193–4201

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Separation and Purification of Schisandrin B from Fructus Schisandrae Ka F. Luk,† Kam M. Ko,‡ and Ka M. Ng*,§ Bioengineering Graduate Program, Department of Biochemistry, and, Department of Chemical Engineering, The Hong Kong UniVersity of Science and Technology, Clear Water Bay, Hong Kong

Schisandrin B (Sch B) is the most abundant and biologically active dibenzocyclooctadiene lignan present in Fructus Schisandrae (FS). It is a highly desirable ingredient for a dietary supplement. While Sch B has been isolated from FS in the laboratory, the experimental procedure was usually lengthy and the yield was relatively low. The objective of this study is to develop a process that is amenable to large-scale recovery of Sch B from FS. The process starts with solid–liquid extraction of FS by petroleum ether to yield a Sch B-containing extract, which is then purified by removing the relatively nonpolar substances, mostly fatty oils, by an adsorption–desorption step. This is followed by a chromatographic process to isolate the Sch B-enriched fraction that is then used to yield Sch B by crystallization. Introduction Schisandrin B (Sch B) (Figure 1a) is the most abundant, biologically active dibenzocyclooctadiene lignan found in Fructus Schisandrae (FS) or Bei Wu-Wei-Zi, a commonly used herb in traditional Chinese medicine. FS is the fruit of Schisandra chinensis (Turcz.) Baillon, a plant that grows in the eastern parts of Russia, North-Eastern China, Korea, and Japan.1 In addition to Sch B, other dibenzocyclooctadiene lignans are also present in FS, including schisandrin A (Figure 1b), schisandrin C (Figure 1c), schisandrol A (Figure 1d), and schisandrol B (Figure 1e). The amount of total lignans in the plant is around 2.2–4.2%, depending on the harvest year and growth location.2,3 FS also contains essential oils, organic acids, and fatty oils.4 Table 1 summarizes the major classes of chemicals and their relative composition in FS. Dibenzocyclooctadiene lignans, which possess a biaryl axis in the dibenzocyclooctadiene structure, can be structurally categorized into two groups, namely, (M)- or (aR) configuration and (P)- or (aS) configuration. In addition, the difference in the substitution pattern of the biaryl unit in Sch B, notably one with three methoxy groups whereas the other one with a methoxy group and a methylenedioxy group, implies that there are four possible stereoisomers for Sch B. Since 1970s, considerable effort has been spent to elucidate the structure of dibenzocyclooctadiene lignans, and the four stereoisomers of Sch B are named (+)γ-schisandrin (or isokadsuranin),5,6 (-)γ-schisandrin,5 kadsuranin,7 and Gomisin N (or (-)Sch B)8,9 (Figure 2). Sch B present in FS is primarily a mixture of (()γ-schisandrin and Gomisin N,3 and the weight ratio of (()γ-schisandrin and Gomisin N is around 2:8 as revealed by reverse phase HPLC. Sch B, as referred to in this article, refers to the total amount of (()γ-schisandrin and (-)Sch B. In the past two decades, the pharmacological activities of Sch B have been extensively investigated. There is a growing body of evidence suggesting its health benefits in the prevention and treatment of a number of diseases.10 Sch B pretreatment has been shown to produce liver and heart protection against oxidative damage by enhancing mitochondrial glutathione antioxidant status and tissue heat shock protein (Hsp)25 and * To whom correspondence should be addressed. Tel.: 852 23587238. Fax: 852 23580054. E-mail: [email protected]. † Bioengineering Graduate Program. ‡ Department of Biochemistry. § Department of Chemical Engineering.

Hsp70 expression.11–15 Sch B treatment was also found to protect against oxidative brain damage in mice16 and attenuate glutamate-induced neurotoxicity in primary cultures of rat cortical neurons.17 A recent study has shown that Sch B could induce apoptosis by down-regulating Hsp70 and up-regulating caspase-3. In addition, Sch B was found to inhibit P-glycoprotein and reverse the multiple drug resistance in cancer cells.18,19 Taken all of these together, Sch B, which apparently offers

Figure 1. Chemical structures of major dibenzocyclooctadiene lignans in FS: (a) Schisandrin B. (b) Schisandrin A. (c) Schisandrin C. (d) Schisandrol A. (e) Schisandrol B.

10.1021/ie071317b CCC: $40.75  2008 American Chemical Society Published on Web 05/15/2008

4194 Ind. Eng. Chem. Res., Vol. 47, No. 12, 2008 Table 1. Major Classes of Chemicals and Their Relative Composition in FS classes of chemicals

examples

dibenzocyclooctadiene lignans fatty oils

Sch A, Sch B, Sch C, schisandrol A, schisandrol B linoleic acid, oleic acid, lauric acid citric acid, malic acid, tartaric acid, ascorbic acid citral, β-chamigrene, β-chamigrenol

organic acids essential oils

relative composition ∼2.2–4.2% ∼38.3% ∼19.9% ∼2.3%

protection for normal tissues but enhances the death of cancer cells, may be used as adjuvant for cancer chemotherapy. Several methods have been reported to isolate Sch B from the lignan-enriched extract of FS.20–22 However, these laboratory procedures usually required lengthy steps and the yield of Sch B was relatively low. Thus, the objective of this research is to synthesize a process for the separation and purification of Sch B from FS that is suitable for large scale manufacturing. Some of the key process parameters will be determined. In addition, there is a great deal interest in expediting the development of processes for the manufacture of natural products.23 The interactions between experiments and synthesis are illustrated in this study as well. Preliminary Conceptual Design A conceptual design consisting of four steps for the manufacture of Sch B has been generated (Figure 3) following a systematic approach for the development of phytochemical manufacturing processes.23 The approach leads the user through the synthesis exercise in a step-by-step manner and provides heuristics, design equations, and general flowsheet structures for decision-making. Because all harvested plants contain a large number of compounds, the recovery of the target compound often goes through a number of purification steps. Thus, in the first step of the process, solid–liquid extraction removes the desired compounds from the solid matrix of FS. Regardless of which solvent is used for extraction, some undesired compounds

Figure 3. Four-step process for the manufacture of Sch B from FS.

in the FS are also coextracted. These include dibenzocyclooctadiene lignans other than Sch B and fatty oils. The second step of the process therefore involves the removal of the relatively nonpolar molecules, mostly fatty oils, in the extract by an adsorption–desorption step. In addition, dibenzocyclooctadiene lignan molecules also differ in polarity because of the presence of different functional groups in the structure. The third step of the process isolates the Sch B-enriched fraction from other dibenzocyclooctadiene lignans by chromatography. Finally, pure Sch B is obtained from the Sch B-enriched fraction by crystallization. Materials and Characterization Chemicals and Herbs. All chemicals used were of analytical grade. Silica gel (0.063–0.200 mm) for adsorption–desorption and column chromatography was obtained from Merck. FS from China was authenticated and supplied by an herbal dealer (Lee Hong Kee Ltd.) in Hong Kong. HPLC Analysis. All HPLC analysis was performed on a Waters Alliance HPLC system which is comprised of a Waters 2956 separation module and a Waters 2996 photodiode array detector. 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) 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. Thin-Layer Chromatography (TLC) Analysis. All TLC analysis was performed on TLC aluminum sheets (Silica Gel 60 F254) from Merck (Darmstadt, Germany), under a solvent system of toluene and ethyl acetate (95:5, v/v). The analytes on TLC sheets were then detected by UV 254 under a fluorescent background using Sch B as reference. Bench Tests

Figure 2. Chemical structures of stereoisomers of Sch B: (a) (+)γSchisandrin or isokadsuranin. (b) (-)γ-Schisandrin. (c) Kadsuranin. (d) Gomisin N or (-)Sch B.

Extraction of FS. FS was dried by heating in a vacuum oven at 50 °C overnight, and then ground into powder; with sieving, the particle size was reduced to less than 1 mm. Extraction was carried out in a double jacketed vessel with stirring, using 100 g of FS powder and 300 mL of the extraction solvent (petroleum ether, acetone or methanol). The extraction temperature of 30 °C was kept constant using a water circulator. It was set as

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Figure 4. Solvent extraction of FS at 30 °C: concentration of Sch B in the extract as a function of time.

Figure 5. Solvent extraction of FS. (a) Effect of solvent-to-solid ratio on the extraction yield of Sch B from FS at 30 °C. (b) HPLC chromatogram of FS petroleum ether extract.

high as possible but without substantial vaporization of the extraction solvent. Liquid samples were taken at different times and the Sch B concentration in the liquid phase was measured by HPLC. Solubility Measurement of Sch B. The solubility of Sch B in different solvents (petroleum ether, acetone and methanol) was measured by the method of isothermal solid-disappearance,24 which determined the amount of solvent required to dissolve a fixed amount of Sch B at 30 °C. Adsorption Isotherm Measurement of Sch B on Silica Gel. The adsorption equilibrium isotherm of Sch B from FS extract on silica gel at 25 °C was measured in a batch system. A volume of 20 mL of different initial concentrations of FS extract in petroleum ether was mixed with 5 g of silica gel. The mixture was shaken in a shaker (Gerbardt, Germany) to establish equilibrium. Liquid samples were taken after 3 days

and the Sch B concentration in the liquid phase was measured by HPLC. The mass of Sch B in the solid phase was determined by mass balance. Adsorption and Desorption Tests for Removal of Relatively Nonpolar Substances. FS extract (200 g) was dissolved in 200 mL of petroleum ether and injected into a preparative silica gel column (200 g, 4.5 cm × 30 cm). The column was eluted with petroleum ether at a flow rate of around 5 mL/min, with the effluent being monitored by TLC. The effluent prior to the appearance of Sch B was pooled and concentrated under reduced pressure in a rotary evaporator to obtain the fatty oil-enriched fraction. Immediately after the appearance of Sch B, the eluting solvent was changed to acetone. At this point, the silica gel in the column appeared as yellowish. The effluent was pooled and concentrated to obtain the lignanenriched fraction. Desorption was stopped when the silica gel

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Figure 6. Adsorption and desorption process. (a) HPLC chromatogram of fatty oil-enriched fraction (F1). (b) HPLC chromatogram of lignan-enriched fraction (F2).

turned to white. Mass balance showed that all the FS extract was completely removed by acetone. Chromatography for Isolation of Sch B-Enriched Fraction. The lignan-enriched fraction (20 g) was dissolved in 20 mL of petroleum ether/acetone (95:5, v/v) and the solution was injected into a preparative silica gel column (200 g, 4.5 cm × 30 cm). The column was eluted with petroleum ether/acetone (95:5, v/v) at a flow rate of around 5 mL/min, with the effluent being monitored by TLC. The effluent fractions showing a single spot of Sch B were pooled and concentrated to obtain the Sch B-enriched fraction. Crystallization of Sch B. The Sch B-enriched fraction was dissolved in hexane (1 g of Sch B fraction to 2 mL of solvent) and was kept at -20 °C overnight. Sch B crystals were then separated from the mother liquor by vacuum filtration, followed by washing with cold hexane. Results and Discussions Step 1. Solvent Extraction of FS. A. Extraction Yield and Rate. The solubility of Sch B in petroleum ether, acetone and methanol at 30 °C was found to be 8.4, 101.8, and 27.3 mg/mL solvent, respectively. While Sch B has the highest solubility in acetone, the time-course of the solid–liquid extraction of FS indicated that the mass of Sch B in the three solvents increased rapidly in the first ten minutes, and then leveled off within an hour (Figure 4). This observation can be explained by the fact that the FS powder of small particle size

(less than 1 mm) enabled the rapid diffusion of Sch B into the liquid phase during the extraction process. The final concentration of Sch B achievable within an hour in the three solvents was similar. Simple material balance shows that practically all the Sch B in the FS powder was extracted. Also, although the solubility of Sch B in petroleum ether is less than those in methanol and acetone, the Sch B concentration in the petroleum ether extract (∼1.1 mg/mL) was still far from the saturation value of 8.4 mg/mL. The solubility effect did manifest itself at the beginning of the extraction process. The higher solubility of Sch B in acetone than in the other two solvents resulted in a slightly higher concentration of Sch B in the extract. Thus, all three solvents are equivalent in terms of extraction yield and rate. B. Extraction Selectivity. The total mass of extract in petroleum ether, acetone and methanol at 30 °C was found to be 11.7, 14.3, and 12.8 g, respectively, by evaporating all the solvent. Since all three solvents yielded an almost identical amount of Sch B from FS and the total mass of extract from petroleum ether extraction was the lowest, this indicates that the smallest amount of unwanted substances was present in the petroleum ether extract. Thus, petroleum ether is the most selective solvent among the three being considered. This agrees well with the report that nonpolar solvents are generally more selective than polar solvents for the extraction of plant secondary products.25 As such, petroleum ether was chosen as the extraction solvent to recover Sch B from FS in this study.

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Figure 7. Effect of feed loading in adsorption and desorption process. (a) Adsorption isotherm of Sch B from FS extract on silica gel at 25 °C. (b) Effect of feed loading on the performance of removal of unwanted impurities.

C. Solvent-to-Solid Ratio in Extraction. To investigate the effect of solvent-to-solid ratio on the extraction yield, 100 g of FS powder was extracted by 300, 600, and 900 mL of petroleum ether for one hour. Figure 5a shows the effect of solvent-tosolid ratio on the extraction yield of Sch B. The data indicated that the extraction yield changed little with an increasing amount of solvent. However, since 100 g of FS already occupies a volume of more than around 250 mL, it is suggested that a minimum of 300 mL of solvent be used to ensure good physical contact between the solvent and 100 g of powdered FS. Figure 5b is the HPLC chromatogram of FS petroleum ether extract which clearly shows the peaks of (()γ-schisandrin and (-)Sch B. However, the presence of other peaks in the chromatogram reveals that other undesired compounds were also

present in the Sch B-containing extract. Therefore, purification steps are necessary to remove these unwanted impurities. Step 2. Removal of Nonpolar Substances by Adsorption and Desorption. A. Fatty Oil-Enriched and Lignan-Enriched Fractions. Other than Sch B, some unwanted compounds such as other dibenzocyclooctadiene lignans and fatty oils were also extracted by the solid–liquid extraction of FS. An adsorption and desorption step with an operation principle similar to that of the frontal chromatographic technique was adopted to remove most of the fatty oils in the petroleum ether extract.26 First, the FS extract was loaded continuously onto the silica gel column packed in petroleum ether. When most of the adsorption sites in the adsorbent were occupied by the retained components (including Sch B), the breakthrough of Sch B occurred (as

4198 Ind. Eng. Chem. Res., Vol. 47, No. 12, 2008 Table 2. Effect of Feed Loading in Adsorption and Desorption Process FS extract-to-silica gel ratio FS extract loaded (g) mass of fatty oil-enriched fraction (g) mass of lignan-enriched fraction (g)

0.5

0.75

1

1.25

1.5

100 51.6

150 96.2

200 145.7

250 137.6

300 97.0

46.4

53.6

53.1

107.2

192.7

detected by TLC). Acetone was then applied for desorption, yielding the lignan-enriched fraction. As evidenced by HPLC analysis, only a minute amount of Sch B was detected in the fatty oil-enriched fraction (Figure 6a). On the other hand, Sch B was mostly retained in the lignan-enriched fraction (Figure 6b). B. Feed Loading in Adsorption and Desorption Process. It is necessary to determine the amount of petroleum ether extract to be loaded to the column. Figure 7a shows the adsorption equilibrium isotherm of Sch B from FS on silica gel at 25 °C. An additional run with a longer shaking time (4 days) was also performed using the same initial concentrations of FS extract in petroleum ether. Identical results were obtained, indicating that 3 days of shaking was sufficient to achieve equilibrium for this system. The concentration of Sch B in FS extract was around 2.8 wt %, and the FS extract was diluted one fold prior to the adsorption process (i.e., 1 g of FS extract was dissolved in 1 mL of petroleum ether). If we further assume a density of 1 g/mL for the FS extract, the Sch B concentration in the feed to the adsorption column can be calculated as follows. Sch B concentration in the feed ) mg Sch B 28 g FS extract ) 14 mg/mL mL mL +1 1 g FS extract g FS extract The adsorption capacity of Sch B on silica gel at this concentration can be estimated by extrapolation (Figure 7a) to be 34 mg/g silica gel. The corresponding adsorption capacity of FS extract on silica gel can be calculated. Adsorption capacity of FS extract on silica gel ) mg Sch B 34 g silica gel g FS extract ) 1.21 g silica gel mg Sch B 28 g FS extract This provides an estimate of the FS extract-to-silica gel ratio that should be applied in the adsorption and desorption step. C. Bench Scale Column Performance. Bench-scale experiments were performed in a silica gel column (200 g, 4.5 cm × 30 cm) to investigate the effect of FS extract-to-silica gel ratio on the effectiveness of the adsorption and desorption process. Table 2 summarizes the effect of feed loading on the mass of both fatty-oil enriched fraction (F1) and lignan-enriched fraction (F2). The percentage of unwanted impurities removed was estimated as (mass of F1/mass of loaded FS extract) × 100%. As shown in Figure 7b, as the FS extract-to-silica gel ratio was increased from 0.5 to 1, the percentage of unwanted impurities removed increased from 51.6% to 72.9%. As the FS extractto-silica gel ratio was further increased to 1.5, the percentage decreased to 32.3%. Thus, for a given amount of adsorbent, there exists an optimum loading of FS extract that offers the highest proportion of unwanted impurities, which is a desirable attribute of the process for the purification of Sch B. The optimum FS extract-to-silica gel ratio (1 g FS extract /g silica

Figure 8. Chromatography process: TLC chromatogram of fractions detected by UV 254.

gel) is less than the adsorption capacity determined from the adsorption isotherm (1.21 g FS extract/g silica gel). This is not surprising because flow dispersion could adversely affect the ideal adsorption capacity obtained under plug flow. Nonetheless, the adsorption isotherm provides a useful estimate of the FS extract-to-silica gel ratio in the adsorption and desorption step. Although more than 70% of the Sch B-containing extract containing primarily fatty oils had been removed, HPLC analysis of the lignan-enriched fraction still revealed the presence of undesired compounds, most notably other dibenzocyclooctadiene lignans (Figure 6b). Thus, the next purification step is to isolate Sch B from other unwanted lignans. Step 3. Chromatographic Separation of Sch B-Enriched Fraction. After injecting the diluted 20 g of lignan-enriched fraction, the silica gel column was eluted by petroleum ether/ acetone (95:5, v/v), with the effluent being monitored by TLC. A Sch B-enriched fraction (∼2.4 g) which was not coeluted with Sch C or Sch A was obtained. Figure 8 shows the TLC chromatogram with six fractions plus the Sch B standard. Sch C would be eluted out first followed by Sch B, Sch A, and other lignans. While the Sch B-enriched fraction obtained from the chromatographic step only showed a single spot of Sch B under UV irradiation in TLC (fraction 4), it could still contain other non-UV-absorbing impurities, which could be detected by charring with concentrated H2SO4. The next step is to remove these impurities to obtain Sch B as the final product. Step 4. Crystallization of Sch B. The crystallization process using 2.4 g of Sch B-enriched fraction yielded 0.5 g of Sch B in solid form. HPLC analysis of the crystallized Sch B showed the presence of highly pure Sch B with (()γ-schisandrin and (-)Sch B in a ratio of 16.7:83.3 (w/w) (Figure 9). Conceptual Process Flowsheet After performing various bench-scale experiments for the proposed process in Figure 3, a conceptual process flowsheet for the manufacture of Sch B from FS can be constructed (Figure 10). The process starts with the extraction of FS by petroleum ether. After filtering out the solid residue (stream 6), if desirable, some of the petroleum ether is recovered in an evaporator (S) and recycled. The FS extract is fed into the silica gel column in the adsorption–desorption step. After feeding the sample, the column is eluted with petroleum ether until Sch B breakthrough occurs. The effluent collected up to this stage, F1, is the fatty oil-enriched fraction. Starting from acetone elution, the effluent

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Figure 9. Crystallization of Sch B: HPLC chromatogram of crystallized Sch B.

Figure 10. Conceptual process flowsheet for the manufacture of Sch B from FS.

collected, F2, is the lignan-enriched fraction. Most of the solvents contained in F1 and F2 can be recovered by evaporation and recycled. Additional amounts of solvents (streams 10 and 11) are required to make up for any loss from this step. The lignan-enriched fraction (F2) would be further processed to obtain Sch B according to a systematic procedure for synthesizing chromatography-crystallization hybrid processes.27 The feed (lignan-enriched fraction) to the column is dissolved in the eluent (petroleum ether/acetone (95:5, v/v)). The column is continuously eluted by the same solvent system and seven fractions, comprising of a fraction of pure solvent and the six fractions shown in the TLC chromatogram (Figure 8), are collected. Fraction 1 containing eluent solvent only is sent back to the eluent stream of the chromatographic column. Sch A,

Sch B, and Sch C are collected separately into fractions 2, 4, and 6, respectively. Fractions 3 and 5 containing Sch B as well as Sch C and Sch A, respectively, are recycled back to the feed stream. Relatively more polar lignans, such as schisandrol A and schisandrol B, are collected in fraction 7. Solvent recovery is carried out in various evaporators (S) and all are recycled back to the chromatographic column. The Sch B-enriched fraction is then further processed to obtain Sch B in solid form. The feed (Sch B-enriched fraction) to the crystallizer is dissolved in hexane. After crystallization, Sch B is recovered as a solid product in stream 47. Hexane in the mother liquor can be largely recovered by evaporator (S) and recycled. Because a significant amount of Sch B remains dissolved in the mother liquor, part of the bottom stream from

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that by petroleum ether. More importantly, supercritical carbon dioxide extraction–offers an FS extract with less unwanted impurities.28 Therefore, SFE might be another feasible product recovery technique instead of solid–liquid extraction. Other process alternatives using different physical properties of the compounds of interest to replace the adsorption–desorption step can also be conceived. For example, since the molecules of fatty oils and dibenzocyclooctadiene lignans differ in polarities, liquid–liquid extraction might be a feasible alternative instead of the adsorption–desorption step. A partially miscible solvent with petroleum ether can be used to extract the desired dibenzocyclooctadiene lignans (including Sch B) from the Sch B-containing extract. As it is expected that the liquid–liquid extraction solvent is polar in nature, the reversedphase chromatography should be used instead of the normalphase to isolate the Sch B-enriched fraction from other lignans in the subsequent chromatography step.

Table 3. Input Information for Material Balance Calculations General Information annual production rate operating hours

10 kg 8000 h/y Solid–Liquid Extraction

solvent-to-FS ratio Sch B concentration in extract

3:1 (mL/g) 2.8 wt %

Adsorption–Desorption impurities removal petroleum ether-to-feed ratio (in mass) (expt data) acetone-to-feed ratio (expt data)

72.9% 4 3

Chromatography eluent-to-feed ratio (in mass) recovery of Sch B in Sch B-enriched fraction Sch B concentration in Sch B-enriched fraction (expt data)

100 75% 50%

Crystallization hexane-to-feed ratio recovery of Sch B in crystal form purge ratio

2:1 (mL/g) 40% 30%

Conclusions

Evaporation assumed solvent recovery in evaporators

A conceptual design for the isolation and purification of Sch B has been developed. First, solid–liquid extraction of FS by petroleum ether yields a Sch B-containing extract. Then, the relatively nonpolar molecules are removed by the adsorption– desorption step, which is followed by a chromatographic process to isolate the Sch B-enriched fraction. Finally, the purified Sch B is recovered from the Sch B-enriched fraction by crystallization. With the material balances, the capital and operating costs can now be estimated. If the manufacturing cost meets the financial expectations, this conceptual design (Figure 10) can be further developed. For example, only Sch B was considered in the adsorption–desorption process. While this is sufficient for synthesizing a process to separate the FS extract into the fatty oil-enriched and lignan-enriched fractions, it is highly desirable to account for the intrinsic multicomponent nature of the system. For the crystallization step, only cooling crystallization was considered. In order to improve the yield, evaporative crystallization should be investigated. Also, crystallization experiments should be repeated with a feed that combines the Sch B-enriched fraction from the chromatographic step and the recycle stream (stream 53). Obviously, a batch plant will be used to manufacture 10 kg of Sch B per year. The batch schedule needs to be formulated to firm up the operating cost. If the manufacturing cost does

98%

the evaporator can be recycled. It may be directed to the dissolver or the chromatography section of the plant. The reminder of this bottom stream is purged in part to avoid accumulation of impurities in the system. Material balance calculations of the process flowsheet can now be performed using the experimental results such as extraction solvent-to-FS ratio and Sch B concentration in extract as well as other input information (Table 3). Table 4 is a stream table which summarizes the flow rates of all input and output streams in the process for a production rate of 10 kg Sch B per year as the base case. For this production rate, input streams of 549.24 g/h of FS, 71.32 g/h of petroleum ether, 6.76 g/h of acetone, and 0.21 g/h of hexane are needed. Other Process Alternatives The process flowsheet presented in this article is not the only option for the separation and purification of Sch B from FS. For example, the use of supercritical fluid extraction (SFE) has recently gained increasing popularity in phytochemical manufacturing processes. Research has shown that extraction of Sch B from FS by supercritical carbon dioxide was comparable to

Table 4. Stream Table for Input and Output Streams for an Annual Production Rate of 10 kg Sch B flow rate (g/h) component

stream 1

stream 2

Sch B soluble impurities insoluble residue petroleum ether acetone hexane

0 0 0 21.42 0 0

1.81 62.45 484.98 0 0 0

stream 10

stream 11

stream 22

stream 43

0 0 0 0 3.86 0

0 0 0 44.76 2.90 0

0 0 0 0 0 0.21

Input Streams 0 0 0 5.14 0 0

flow rate (g/h) component

stream 6

stream 20

stream 26

Sch B soluble impurities insoluble residue petroleum ether acetone hexane

0 0 484.98 0 0 0

0 45.53 0 0 0 0

0 1.47 0 0 0 0

stream 34

stream 37

stream 47

stream 52

solvent loss

0 13.04 0 0 0 0

1.25 0 0 0 0 0

0.56 1.47 0 0 0 0

0 0 0 71.33 6.76 0.21

Output Streams 0 0.95 0 0 0 0

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not meet the financial expectations, other process alternatives such as liquid–liquid extraction and supercritical extraction for replacing the adsorption–desorption step should be considered. Given that the Sch B preparation contains both (-)Sch B and (()γ-schisandrin and that (-)Sch B was found to be more potent than (()γ-schisandrin in enhancing cellular glutathione and protecting against oxidative injury in cultured cardiomyocytes and hepatocytes,29,30 it is desirable to recover (-)Sch B in a purified form for pharmaceutical application. Process design for the production of (-)Sch B from Sch B is underway. Acknowledgment Research support of the Research Grants Council (HKUST618207) is gratefully acknowledged. Literature Cited (1) Lebedev, A. A. Limonnik; Medicinal Publishing House: Tashkent, Uzbek SSR, 1971. (2) Slanina, J.; Taborska, E.; Lojkova, L. Lignans in the seeds and fruits of Schisandra chinensis cultured in Europe. Planta Med. 1997, 63, 277. (3) Nakajima, K.; Taguchi, H.; Ikeya, Y.; Endo, T.; Yosioka, I. The constituents of Schizandra chinensis Baill. XIII. Quantitative analysis of lignans in the fruits of Schizandra chinensis Baill. by high performance liquid chromatography. Yakugaku Zasshi 1983, 103 (7), 743. (4) Zhou, R. Resource Science of Chinese Medicinal Materials; Chinese Medicine Science and Technology Press: Beijing, China, 1993. (5) Ikeya, Y.; Taguchi, H.; Yosioka, I. The constituents of Schizandra chinensis Baill. X. The structures of γ-schizandrin and four new lignans, (-)-gomisins L 1, and L 2, (()-Gomisin M 1 and (+)-Gomisin M 2. Chem. Pharm. Bull. 1982, 30, 132. (6) Li, L.; Xue, H.; Tan, R. Dibenzocyclooctadiene lignans from roots and stems of Kadsura coccinea. Planta Med. 1985, 51, 297. (7) Tan, R.; Li, L.; Fang, Q. Studies on the chemical constituents of Kadsura longipedunculata: Isolation and structure elucidation of five new lignans. Planta Med. 1984, 50, 414. (8) Ikeya, Y.; Taguchi, H.; Yosioka, I.; Kobayashi, H. The constituents of Schizandra chinensis Baill. V. The structures of four new lignans, Gomisin N, Gomisin O, epigomisin O and Gomisin E, and transformation of Gomisin N to deangeloylgomisin B. Chem. Pharm. Bull. 1979, 27, 2695. (9) Ko, K. M.; Poon, M. K. T.; Ip, S. P.; Wu, K. Protection against carbon tetrachloride liver toxicity by enantiomers of schisandrin B associated with differential changes in hepatic glutathione antioxidant system in mice. Pharmaceut. Biol. 2002, 40, 298. (10) Ko, K. M.; Mak, H. F. Schisandrin B and other Dibenzocyclooctadiene lignans. In Herbal and Traditional Medicine: Molecular Aspects of Health; Packer, L., Halliwell, B., Ong, C. N., Eds.; Marcel Dekker: New York, Basel, Hong Kong, 2004; p 289. (11) 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. (12) Ip, S. P.; Yiu, H. Y.; Ko, K. M. Schisandrin B protects against menadione-induced hepatotoxicity by enhancing DT-diaphorase activity. Mol. Cell. Biochem. 2000, 208, 151. (13) Ip, S. P.; Poon, M. K. T.; Che, C. T.; Ng, K. H.; Kong, Y. C.; Ko, K. M. Schisandrin B protects against carbon tetrachloride toxicity by

enhancing the mitochondrial glutathione redox status in mouse liver. Free Radic. Biol. Med. 1996, 21, 709. (14) 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. (15) Chiu, P. Y.; Ko, K. M. Schisandrin B protects myocardial ischemiareperfusion injury partly by inducing Hsp25 and Hsp70 expression in rats. Mol. Cell. Biochem. 2004, 266, 139. (16) Ko, K. M.; Lam, B. Y. H. Schisandrin B protects against tert-butylhydroperoxide induced cerebral toxicity by enhancing glutathione antioxidant status in mouse brain. Mol. Cell. Biochem. 2002, 238, 181. (17) Kim, S. R.; Lee, M. K.; Koo, K. A.; Kim, S. H.; Sung, S. H.; Lee, N. G.; Markelonis, G. J.; Oh, T. H.; Yang, J. H.; Kim, Y. C. Dibenzocyclooctadiene lignans from Schisandra chinensis protect primary cultures of rat cortical cells from glutamate-induced toxicity. J. Neurosci. 2004, 76, 397. (18) Qiangrong, P.; Wang, T.; Lu, Q.; Hu, X. Schisandrin B - a novel inhibitor of P-glycoprotein. Biochem. Biophy. Res. Commun. 2005, 355, 406. (19) Li, L.; Lu, Q.; Shen, Y.; Hu, X. Schisandrin B enhances doxorubicin-induced apoptosis of cancer cells but not normal cells. Biochem. Pharmocol. 2006, 71, 584. (20) Chen, Y.; Shu, Z.; Li, L. Studies on Fructus Schizandrae IV. Isolation and determination of the active compounds (in lowering high SGPT levels) of Schizandra chinensis Baill. Scientia Sinica 1976, 19 (2), 276. (21) Ikeya, Y.; Taguchi, H.; Yosioka, I.; Kobayashi, H. The constituents of Schizandra chinensis Baill. I. Isolation and structure determination of five lignans, Gomisin A, B, C, F and G, and the absolute structure of schizandrin. Chem. Pharm. Bull. 1979, 27, 1383. (22) Huang, T.; Shen, P.; Shen, Y. Preparative separation and purification of deoxyschisandrin and γ-schisandrin from Schisandra chinensis (Turcz.) Baill by high-speed counter-current chromatography. J. Chromatogr. A 2005, 1066, 239. (23) Harjo, B.; Wibowo, C.; Ng, K. M. Development of natural product manufacturing processes: Phytochemicals. Chem. Eng. Res. Des. 2004, 82 (A8), 1010. (24) Kwok, K. S.; Chan, H. C.; Chan, C. K.; Ng, K. M. Experimental determination of solid-liquid equilibrium phase diagrams for crystallizationbased process synthesis. Ind. Eng. Chem. Res. 2005, 44 (10), 3788. (25) Walton, N. J.; Brown, D. E. Chemicals from plants: perspectiVe on plant secondary products; Imperial College Press: London, 1999. (26) Hill, D. A.; Mace, P.; Moore, D. Frontal chromatographic techniques in preparative chromatography. J. Chromatogr. 1990, 523, 11. (27) Fung, K. Y.; Ng, K. M.; Wibowo, C. Synthesis of ChromatographyCrystallization Hybrid Separation Processes. Ind. Eng. Chem. Res. 2005, 44, 910. (28) Lojkova, L.; Slanina, J.; Mikesova, M.; Taborska, E.; Vejrosta, J. Supercritical fluid extraction of lignans from seeds and leaves of Schizandra chinensis. Phytochem. Anal. 1997, 8, 261. (29) Chiu, P. Y.; Leung, H. Y.; Poon, M. K. T.; Mak, D. H. F.; Ko, K. M. (-)Schisandrin B is more potent than its enantiomer in enhancing cellular glutathione and heat shock protein production as well as protecting against oxidant injury in H9c2 cardiomyocytes. Mol. Cell. Biochem. 2006, 289, 185. (30) Chiu, P. Y.; Leung, H. Y.; Poon, M. K. T.; Mak, D. H. F.; Ko, K. M. Effects of schisandrin B enantiomers on cellular glutathione and menadione toxicity in AML12 hepatocytes. Pharmacology 2006, 77, 63.

ReceiVed for reView September 29, 2007 ReVised manuscript receiVed February 22, 2008 Accepted March 6, 2008 IE071317B