Supercritical Fluid Science and Technology - American Chemical

Chapter 26

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on September 16, 2015 | http://pubs.acs.org Publication Date: August 29, 1989 | doi: 10.1021/bk-1989-0406.ch026

Extraction and Isolation of Chemotherapeutic Pyrrolizidine Alkaloids from Plant Substrates Novel Process Using Supercritical Fluids 1

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Steven T. Schaeffer , Leon H. Zalkow , and Amyn S. Teja School of Chemical Engineering, Georgia Institute of Technology, Atlanta, GA 30332-0100

Pyrrolizidine alkaloids have long been known to be antitumor active and, more recently, have become of interest as anti-cancer agents. They occur naturally in several plant species, but are often difficult to extract and isolate from the plant material without degradation or the use of toxic solvents. The extraction of a model pyrrolizidine alkaloid, monocrotaline, from the seeds of Crotalaria spectabilis was investigated in this work. The crushed seeds of Crotalaria spectabilis were first contacted with supercritical carbon dioxide and, as expected, the oils compris ing the bulk of the seed material were preferentially extracted. The addition of ethanol and water as co-solvents in the fluid phase led to the appearance of monocrotaline in the extract. Monocrotaline contents as high as 24% of the total extract could be obtained with carbon dioxide-ethanol mixtures. In order to increase the extract purity further, two additional pro cesses were developed. The temperature-solubility cross-over point was utilized to obtain extracts containing as much as 50% monocro taline. A second novel process incorporating ion exchange resins was also studied and yielded extracts containing 94 to 100% monocro taline. Because this technique depends only on the basic character common to the pyrrolizidine alkaloids, it is expected to be equally effective in the extraction and isolation of the other members of this class and, in fact, could be extended to the other classes of basic alkaloids. Current address: Ε. I. du Pont de Nemours and Company, P.O. Box 2042, Wilmington, NC 28402 Current address: School of Chemistry, Georgia Institute of Technology, Atlanta, GA 30332

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0O97-^156/89/0406-O416$O6.00/0 ο 1989 American Chemical Society

In Supercritical Fluid Science and Technology; Johnston, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


26.

SCHAEFFER E T AL.

Pyrrolizidine Alkaloidsfrom Plant Substrates

Supercritical fluid extraction processes are particularly appropriate for the sepa ration and isolation of biochemicals where thermal decomposition, chemical mod ification, and physiologically-active solvents are undesirable. Examples of these bioseparations include the extraction of oils from seeds using carbon dioxide (1), of nicotine from tobacco using carbon dioxide-water mixtures (2), and of caffeine from coffee beans again using carbon dioxide-water mixtures (3). In the present investigation, supercritical carbon dioxide and carbon dioxide + co-solvent mixtures were used to extract and isolate a model pyrrolizidine alkaloid from its parent plant. Pyrrolizidine alkaloids have been used in herbal medicine to combat tumors as long ago as the fourth century A.D. (4) and to treat cancer since the tenth century A.D. (5). More recently, they have received increasing attention as chemotherapeutic drugs. Processes for their separation, however, are specific to each alkaloid, and either lead to chemical modification of the alkaloid or require the use of solvents which must then be completely removed from the extract. The purpose of this study was to develop a general supercritical fluid based process for the separation and purification of pyrrolizidine alkaloids such as monocrotaline (Cieï^NOe, MW=325.3). Monocrotaline was selected as a model because ofitsrole in the development of semisynthetic pyrrolizidine alkaloids (6) and because it occurs in several species of Crotalaria. The seeds of Crotalaria spectabilis served as the source of monocrotaline in this study. Experimental A dual-feed single-pass supercritical fluid extraction apparatus was constructed as shown in Figure 1. Pressurized carbon dioxide was filtered, liquified in an ice bath C, and pumped to the system pressure in an Eldex dual-channel metering pump E. The co-solvent was filtered and fed from a graduated cylinder D into the other head of the pump. The solvents were mixed in a Kenics static mixer I immersed in the constant temperature bath F . The homogeneous mixture then passed through an equilibrium cell packed with alternating layers of solute and glass beads. Equilibrium between the solute and the fluid was achieved by al lowing sufficient fluid residence time in the equilibrium cell. This was confirmed experimentally by monitoring the composition of the supercritical mixture at the exit of the cell. An in-line temperature-controlled Mettler-Parr fluid densiometer L was used to determine the fluid density. The mixture was then depressurized in a heated micrometering valve 0 and the condensables (solute and a portion of the co-solvent) were collected in a collection vessel Q. Condensation was ensured by immersing the collection vessel in an ice bath. The gas mixture then passed through a bank of rotameters R for flow visualization, through a gas sampling valve S for composition analysis, and finally through a wet test meter V for flow totalization. A gas chromatograph U with aflameionization detector was used for analysis of the carbon dioxide + ethanol stream. The tubing outside the bath was heated to the bath temperature to prevent deposition of material in the lines.


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SUPERCRITICAL FLUID SCIENCE A N D T E C H N O L O G Y


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26.

SCHAEFFERETAL.

lyrrolizidine Alkahiasjrom Plant Substrates419

Liquid solvent contained in M was used to flush the micro-metering valve of any deposited material. The system pressure was maintained using a back-pressure regulator J pres surized with high pressure nitrogen from a tank A. The pressure was monitored by two calibrated Heise gauges PI and P2. The bath temperature was controlled by a controller G and was monitored using two calibrated thermocouples T l and T2. The amount of extract in the collection vessel was determined gravimetrically. The amount of monocrotaline in the extract was determined using a proton NMR technique developed by Molyneaux et al. (7). Purity and Preparation of Materials. The carbon dioxide used was Coleman instrument grade with a purity of >99.9%. Pure ethanol (>99.9%) was obtained by reactive distillation of HPLC grade ethanol with magnesium turnings cat alyzed with iodine. Water was obtained by double distillation. Ethanol used for valve and collection vesselflushingwas 95 wt.% ethanol - 5 wt.% water. All sol vents and co-solvents were dried to confirm the absence of solids. Monocrotaline (>99%) was obtained from the seeds of Crotalaria spectabilis using the method described by Gelbaum et al. (6). The seeds of Crotalaria spectabilis were obtained in Clarke County, Georgia in November, 1984. These seeds were analyzed and found to contain 1.9 wt.% monocrotaline and 2.5 wt.% hexane-extractable lipid material. The seeds (5mm indiameter) were milled to 1 mm to break the hard outer coat and to expose the inner seed material containing the monocrotaline. The seed fragments were sieved to remove the 850+ micron fraction (predominantly outer coat fragments) and the 850- micron fraction was packed in the equilibrium cell. Pure Component Studies Before investigating the extraction of monocrotaline from Crotalaria spectabilis, the solubility of pure monocrotaline was measured. This was done to determine the magnitude of the solubility, to evaluate the effect of co-solvents, and to con firm the integrity of the extracted monocrotaline. Carbon Dioxide - Monocrotaline System. The solubility of pure monocrotaline was measured at three temperatures (308.15, 318.15, and 328.15K) at pressures ranging from 8.86 MPa to 27.41 MPa. The solubilities ranged form 6xl0" to 4.4xl0~ mole fraction (Figure 2). Long extraction times were used to reduce the experimental error to lxlO" mole fraction. These low solubilities are typical of large polar biomolecules. 6

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Carbon Dioxide - Ethanol - Monocrotaline System. Since monocrotaline is sol uble in ethanol, ethanol was selected as a co-solvent. The carbon dioxide - ethanol phase diagram of Panagiotopoulos and Reid (8) was used to ensure that no liquid phases were present at any time in the equilibrium cell. This was also confirmed by visual observation. The solubility of monocrotaline in carbon dioxide 4- 5 mol% and 10 mol% ethanol was measured at three temperatures and pressures ranging from 10.34



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SUPERCRITICAL FLUID SCIENCE A N D T E C H N O L O G Y

1E-3 • 308.15K • 318.15K 0 328.15K

•

-O-O1E-4

10%ET0H

1E-4

/

1E-5

1E-6

200

300

400

500 600 700 DENSITY ( K G / M 3 )

0%ET0H

800

900

1000

Figure 2: Solubility of monocrotaline in carbon dioxide, 95 mole% carbon dioxide -f 5 mole% ethanol and 90 mole% carbon dioxide + 10 moie% ethanol.


26.

SCHAEFFERET AL.

Pyrrolizidine AlkaloidsfromPlant Substrates421 4

to 27.41 MPa. The solubility was as high as 2.21xl0"" mole fraction (Figure 2). The largest increase due to the addition of ethanol was 25 fold indicating that ethanol is an effective co-solvent for monocrotaline. Also, it was found that the temperature-solubility cross-over point (dy/dT)p = 0 advanced nearly linearly with ethanol concentration.


Complex Substrate Studies Due to the relative success of the pure component solubility studies, the same series of experiments were carried out using the complex seed material. Three systems were investigated to evaluate the ability of supercritical fluids to ex tract monocrotaline from the seeds of Crotalaria spectabilis. Pure carbon dioxide was studied with the expectation that the oils would be preferentially extracted. Ethanol was added as a co-solvent to increase the solubility of monocrotaline. Also, due to its success in the extraction of caffeine and nicotine, water was used as a co-solvent. Carbon Dioxide - Crotalaria Spectabilis System. The fluid phase concentration was found to be time-dependent (Figure 3). At the start of the extraction, the concentration was constant indicating that it was equal to the equilibrium con centration. After approximately one mass percent of the initial mass of the bed had been removed, however, the exit concentration began to decrease. This result may be explained by a combination of intraparticle diffusion and bulk mass transfer processes. As material is extracted from the exposed areas of the seed, the solvent must travel further through the pores to reach the solute. Also, as the entrance portion of the bed becomes depleted of soluble components, the effective bed length decreases until the residence time is insufficient to achieve equilibrium. Similar effects were observed in seed oil extraction by Fat tori (1) and Taniguchi et al. (9). The solubility of the seed material in carbon dioxide was defined as the intial concentration prior to observation of these depletion effects. The solubility was found to range from 0.016 wt.% to 0.6 wt.% (Figure 4). Analysis of the extracts revealed that the monocrotaline content was very low (

Supercritical Fluid Science and Technology - American Chemical

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