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Recovery of gossypol acetic acid from cottonseed soapstock
Industrial Crops and Products 14 (2001) 113– 123 www.elsevier.com/locate/indcrop
Recovery of gossypol acetic acid from cottonseed soapstock Michael K. Dowd *, Scott M. Pelitire Commodity Utilization Research Unit, Southern Regional Research Center, ARS, USDA, 1100 Robert E. Lee Bl6d., New Orleans, LA 70124 USA Received 20 September 2000; accepted 13 December 2000
Abstract Gossypol is a yellow pigment found in the cotton plant and is of scientific and medical interest because of its anti-tumor, anti-fertility, and anti-viral properties. In order to support additional studies into the medicinal activity of gossypol, methods are needed to isolate large quantities of the compound in high purity. A process is described to recover gossypol from cottonseed soapstock, a low-value co-product of crude oil refining that can contain concentrations of gossypol as high as 8%. Soapstock is refluxed in acidic methyl ethyl ketone (MEK) to hydrolyze covalently bound gossypol. Upon cooling, the mixture separates into organic and aqueous phases with the gossypol distributing between the two phases. The MEK phase is recovered, and the aqueous phase is re-extracted with additional volumes of MEK. After combining and concentrating the MEK extracts, acetic acid is added to induce gossypol crystallization, which precipitates as an equimolar crystalline complex with acetic acid (gossypol acetic acid). From a soapstock sample containing 3.7% gossypol, 63% of the gossypol was recovered as an 87% gossypol acetic acid product. A single re-crystallization of this crude material yielded a 99% gossypol acetic acid product with an overall recovery of 58%. With different soapstock samples, the yield of crude product was positively correlated with the initial gossypol concentration in the soapstock. Recovery, however, was not well correlated with soapstock gossypol concentration, possibly due to the co-extraction of other components that influence the solubility of gossypol in the crystallization solution. Published by Elsevier Science B.V. Keywords: Cottonseed; Extraction; Foots; Gossypol; Gossypol acetic acid; Soapstock
1. Introduction Gossypol [1,1’,6,6’,7,7’-hexahydroxyl-5,5’-diisopropyl-3,3’-dimethyl-(2,2’-binaphthalene)-8,8’-dicarboxaldehyde] is a polyphenolic yellow compound found in pigment glands distributed * Corresponding author. Tel.: + 1-504-2864339; fax: + 1504-2864367. E-mail address: [email protected] (M.K. Dowd).
throughout the cotton plant (Gossypium sp.). The compound has been associated with a wide range of biological and medicinal activity, including anti-tumor (Tso, 1984; Chang et al., 1993; Flack et al., 1993), anti-fertility (Qian and Wang, 1984; Yu, 1987; Cosentino and Matlin, 1997), and antiviral (Lin et al., 1989) effects. Gossypol is also responsible for toxic effects associated with the overuse of cottonseed products in animal feeds (Berardi and Goldblatt, 1969).
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Gossypol exists in a number of tautomeric and isomeric forms. Because of the placement of methyl and hydroxyl groups on the carbon atoms adjacent to gossypol’s binaphthalene bridge (Fig. 1), rotation about this bond is severely restricted. As a consequence, gossypol exhibits atropisomerism and can be found in two enantiomeric forms. The cotton plant produces both forms in a ratio that varies from approximately 60:40 ( + / −) to 45:55 (+/ − ). Several studies indicate that the gossypol isomers contribute disproportionally to the compound’s biological activity, e.g. ( − )-gossypol appears to be responsible for most of the contraceptive effects (Yu, 1987; Lindberg et al., 1987). Because of these differences in activity, there has been interest in developing techniques to separate the (+)- and (− )-gossypol forms. Recently, we reported a crystalline form of gossypol that resolves the enantiomers (Dowd et al., 1999), and we are currently trying to scale up this process to produce the enantiomers in useful amounts. This paper describes a new procedure to prepare the large amounts of gossypol needed to support this work. The earliest isolations of gossypol were from cottonseed soapstock, a by-product of oil refining (Longmore, 1886; Marchlewski, 1899). These initial preparations, however, were quite crude, and the methods were quickly overshadowed by other approaches. Subsequent procedures employed cottonseed or defatted cottonseed (Carruth, 1918), the root bark of the cotton plant (Royce et al., 1941), or isolated pigment glands (Castillon et al., 1948). Pons et al. (1959) proposed using cottonseed gums, a by-product of crude oil refining. At the time, this method was advantageous because gums were highly concentrated in gossypol (4 – 6%) and the use of gums did not interfere with
the utilization of the valuable oil or meal. Unfortunately, the cottonseed industry has subsequently converted from crude oil refining to miscella refining (refining before solvent stripping), and the production of gums has been eliminated. Therefore, an alternative procedure is needed to produce large amounts of research-grade gossypol. Because the concentration of gossypol in cottonseed soapstock can be as high as 8% (Dowd, 1996), a reinvestigation of this material for gossypol recovery is warranted. The general strategy of most recovery methods is to extract gossypol into an organic solvent, concentrate the resulting solution, and precipitate gossypol by adding acetic acid. The product is a crystalline inclusion complex containing an equimolar ratio of gossypol and acetic acid (gossypol acetic acid) (Carruth, 1918). This form of gossypol is relatively stable. It contains the enantiomers in an equimolar ratio, and is widely used as an analytical standard for measuring gossypol in animal tissues and cottonseed products (Hron et al., 1990; Kim et al., 1996; Hron et al., 1999). Methods have been reported to recover pure gossypol from gossypol acetic acid (Carruth, 1918; King and Thurber, 1953; Pons et al., 1959); however, these procedures are tedious and prone to unwanted side reactions. Our process recovers a 99% gossypol acetic acid product from cottonseed soapstock. The process is similar to the method used by Pons et al. (1959) for isolating gossypol acetic acid from cottonseed gums. However, some of the separation steps were modified, and the optimal conditions were different. 2. Material and methods
2.1. Sample preparation and handling
Fig. 1. Structure of gossypol.
Soapstock from a Louisiana miscella-refining cottonseed oil mill was collected immediately downstream of the soapstock centrifuge and cooled rapidly with ice. The National Cottonseed Products Association (Memphis, TN) also provided several additional soapstock samples from different oil mills. All samples were stored at − 20°C until used.
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Fig. 2. Process schematic for the recovery of gossypol from cottonseed soapstock. Process was optimized in four steps (1 – 4) as labeled.
Soapstock gossypol concentration was determined by the AOCS (American Oil Chemists’ Society, 1998) Official Method Ba 8– 78. Alkali concentration was estimated by dispersing 0.5 g of soapstock in 15 ml of deionized filtered water and measuring pH.
2.2. Reco6ery process The procedure (Fig. 2) has five processing operations. These include treating soapstock with acid to hydrolyze bound gossypol, partitioning the gossypol into an organic phase, washing the residual aqueous phase of additional gossypol, concentrating the combined gossypol solution by vacuum distillation, and inducing crystallization by adding acetic acid. For the hydrolysis step, 10091 g of soapstock was weighed into a 1-l flask with 100 ml of methyl ethyl ketone (MEK) containing phosphoric acid and a stir-bar. The flask was connected to a condenser, and the mixture was stirred while heating to reflux. At the end of the hydrolysis step, the flask was cooled to room temperature in an icewater bath, and the contents were transferred to a centrifuge bottle. The mixture was centrifuged at 5000×g for 5 min to separate the emulsion into MEK and aqueous phases. After recovering the
MEK phase, the aqueous phase was contacted with additional 50 ml volumes of MEK. Each MEK/aqueous mixture was briefly shaken by hand and re-centrifuged at 5000× g for 5 min. The combined extracts were filtered through c 4 Whatman paper to remove any residual insoluble material, and a small amount of MEK was used to wash the filter paper. The filtered solution was concentrated with a rotary evaporator using house vacuum ( 25 in Hg vacuum), 5°C condenser water, and 50°C heating water. After transferring the concentrated solution into a graduated cylinder or vial, glacial acetic acid was added to induce crystallization. During the crystallization process, the solution was stored in the dark to prevent photo-oxidation. Crude product was recovered by vacuum filtration through preweighed c 4 Whatman paper, and the gossypol acetic acid precipitate was washed with hexane until the filtrate was colorless. Vacuum was applied until no additional filtrate was collected. The product was stored overnight in a vacuum oven at room temperature to remove residual hexane.
2.3. Process optimization The process was optimized in a series of four experimental studies (Fig. 2) starting with the
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hydrolysis step (Step 1) and working toward the crystallization step (Step 4). Preliminary experiments were conducted to find a reasonable set of starting values for process variables and to bracket the ranges of values needed to ensure that optimal conditions were found. Each step was optimized in sequence carrying over the optimal conditions from the previous steps. Step 1 of the optimization focused on the hydrolysis conditions. Phosphoric acid concentrations of 0.6, 1.0, 1.4, or 1.8 M and reaction times of 0.5, 1.0, 2.0, or 4.0 h were examined. All other steps were kept constant (two 50-ml MEK washings, final concentrate volume of 50 ml per 100 g initial soapstock mass, addition of acetic acid to a 1:3 (v/v) ratio, and crystallization at room temperature for 24 h without agitation). Step 2 tested the effect of including up to five 50-ml MEK washings of the aqueous residue. Step 3 studied concentration conditions (final volume of the MEK –gossypol concentrate and the ratio of MEK-to-acetic acid in the crystallization solution). For this study, the combined gossypol– MEK fractions were concentrated to 40, 50, or 60 ml, and acetic acid was added to give final acetic acid/MEK ratios of 1:10, 1:5, 1:3, or 1:2. Crystallization conditions were studied in Step 4. Time was varied from 0.25 to 48 h with and without agitation. For agitation, a rocker table was used with a 9 20° angle at 90 tilts per minute. Temperature (4°C, room temperature, and 50°C) was also considered in the preliminary experiments used to define starting conditions. To increase the number of crystallization conditions that could be studied, the concentrated gossypol– MEK solutions from four separate 100-g runs were combined and partitioned into 20-ml aliquots.
2.4. Sample purity Purity of crude gossypol products was determined by HPLC against a gossypol acetic acid standard (Kim et al., 1996). Crude product (100 mg) was weighed into a 100-ml volumetric flask and filled to the mark with reagent grade acetonitrile. A 0.5-ml aliquot was transferred to a test tube, mixed with 1.0 ml of a complexing reagent, and heated at 95–100°C for 30 min with the tube
capped. The complexing reagent was 3-amino-1propanol (2 ml), glacial acetic acid (10 ml), and dimethylformamide added to a final volume of 100 ml. After cooling, the derivatized sample was diluted with 9 ml of acetonitrile. The HPLC system consisted of a pumping system and photo-diode array detector (Models 2690 and 996, respectively, Waters Corp., Milford, MA) with Inertsil C18 reverse-phase guard and cartridge columns (SGE, Inc., Austin, TX). The mobile phase was a 0.01 M KH2PO4 buffer adjusted to pH 3 with H3PO4 (20%) in acetonitrile (80%) pumped at a flow rate of 1 ml/min. Injections were 20 ml, and the gossypol complex was detected at 254 nm. A sample of gossypol acetic acid originally isolated and purified from cottonseed gums and believed to be 99% pure was used to prepare a reference standard. This sample was repeatedly re-crystallized by dissolving in acetone (1:6 w/v), filtering with c 4 Whatman paper, and adding one-third volume of acetic acid. The initially dark solutions were shaken until yellow particles were noticeable ( 3 min), and the mixture was stored in the dark for 2 h. Precipitated gossypol acetic acid was isolated by vacuum filtering through c 4 Whatman paper, washing the precipitate with hexane until the filtrate was colorless, and drying under house vacuum overnight at room temperature. The color of the product (bright yellow) improved slightly with each re-crystallization, and the third and final re-crystallized sample was used as the reference material for this work.
2.5. Purification of soapstock gossypol acetic acid The purity of the initial gossypol product was increased by re-crystallization from solutions of MEK and acetic acid. Several samples from the development studies were combined to provide the starting material. For these experiments, 2.090.1 g of crude product was dissolved in MEK (1:7 w/v), filtered through c 4 Whatman paper, and one-half volume of acetic acid was added to induce crystallization. The solutions were agitated in the dark at room temperature for 4 h. Gossypol acetic acid was recovered by filtering through c4 Whatman paper, rinsing with
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hexane until the filtrate was colorless, and vacuum drying overnight at room temperature. The process was repeated up to three times. The initial filtration step was omitted after the first re-crystallization. The purity of the re-crystallized products was determined following the procedure outlined for the crude samples. Testing of the final gossypol acetic acid product was conducted to ensure authenticity. The melting point was determined with a capillary melting apparatus, and the carbon, hydrogen, and nitrogen concentrations were determined by combustion methods. The UV– Vis spectrum was recorded in chloroform with a spectrophotometer (Model UV160 U, Shimadzu Scientific Instruments, Inc., Columbia, MD), and the spectrum of the di-3-amino-1-propanol gossypol complex was determined with the HPLC photo-diode detector.
2.6. Calculations and statistics Crude product yield was calculated as the weight of precipitated product divided by the initial wet weight of soapstock. Purity was measured as the detector area per unit mass of the sample divided by the detector area per unit mass of the standard. Gossypol recovery was calculated as the amount of gossypol in the crude product divided by the amount of gossypol in the soapstock. For the purification study, similar definitions were used except that the yield and recovery values were referenced to the starting crude gossypol product. In addition, a net gossypol recovery was defined to account for the recovery from the initial isolation process and the recovery from purification. All reported values were expressed on a percent basis. Experiments were conducted in triplicate. The SAS GLM analysis of variance procedure (SAS Institute Inc., 1989) was used to test for main and interaction effects on yield, purity, and recovery. In addition, pairwise ‘t’-tests were computed with the MEANS statement (h = 0.05). When two variables were included in the experimental design (Steps 1 and 3), least square means for the interaction effects were also tested.
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3. Results and discussion
3.1. Hydrolysis conditions (Step 1) Acid concentration, reaction time, and the interaction of acid concentration and time all significantly affected crude product yield (PB 0.001) (Table 1). The yield was very low at 0.6 M phosphoric acid, but increased at the higher concentrations. With sufficient acid concentration and hydrolysis time, product yield appeared to reach a ‘plateau’ that represented the maximum yield. Acid concentration also affected product purity (PB0.0001) (Table 1). At low acid concentration (0.6 M), purity varied from 39 to 64% over the reaction times studied. At greater acid concentrations (]1.0 M), product purity was significantly higher (82–87%). Both the low yield and low purity contributed to the low gossypol recovery (B 10%) achieved at low acid concentration. Hydrolysis time was important at the 1.0 M phosphoric acid concentration, but less important at higher acid concentrations (1.4 and 1.8 M). As for product yield, a ‘plateau’ state was reached with sufficient acid and reaction time, which corresponded to the maximum recovery. The strong influence of acid concentration on product yield and recovery confirms that soapstock gossypol is mostly in a bound form (Dowd, 1996). The highest gossypol recovery was 57.09 2.5% with 1.8 M phosphoric acid and a 1-h reaction time. This recovery, however, was not significantly different from the recoveries obtained with 1.8 M acid concentration and other reaction times or the recoveries obtained with a 1.4 M acid concentration and reaction times greater than 1.0 h. To reduce acid use, the optimal reaction conditions were taken to be 1.4 M phosphoric acid and a 2-h hydrolysis period.
3.2. Extraction of aqueous residue (Step 2) Additional MEK washing of the aqueous residual affected the crude product yield, purity, and recovery (P B0.0001). Yield significantly increased with the first three extractions, but did not
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ditional washings beyond the third did not significantly increase gossypol recovery (Table 2). To achieve the highest gossypol purity, only the initial extraction should be conducted. Recovery of gossypol, however, improves with up to three additional washings of the aqueous residue. In this work, our first concern was to achieve high recovery. Therefore, three extractions were con-
increase with the fourth or fifth extraction (Table 2). Product purity decreased with the first three additional washings (Table 2). As with yield, recovery increased with additional washing, but the increases were less pronounced because of the decreasing trend associated with product purity. Three washings increased the recovery 58% over the recovery obtained without the washings. Ad-
Table 1 Effect of hydrolysis conditions on the yield, purity, and recovery of gossypol acetic acid isolated from cottonseed soapstocka Reaction time (h)
Phosphoric acid concentration (M) 0.6
1.0
1.4
1.8
0.5 1.0 2.0 4.0
Crude product yield (%) 0.38 9 0.25 0.299 0.09 0.289 0.21 0.3690.26
1.13 9 0.30 2.00 90.15 2.14 9 0.19 2.24 9 0.26
2.22 9 0.14 2.39 90.08 2.69 9 0.07b 2.51 9 0.08b
2.48 90.13b 2.69 90.26b 2.63 90.11b 2.76 90.20b
0.5 1.0 2.0 4.0
Purity (%) 49.09 36.3 64.8 9 12.6b 39.89 32.0 48.19 36.0
84.9 91.5b 85.8 9 7.1b 86.4 9 3.4b 86.0 94.7b
87.4 92.3b 85.5 9 6.7b 84.9 94.7b 86.2 92.3b
83.2 9 4.2b 87.1 9 5.2b 82.5 96.4b 82.6 9 3.1b
0.5 1.0 2.0 4.0
Reco6ery (%) 6.09 5.1 4.79 2.3 3.7 94.5 5.595.8
23.4 96.1 42.0 9 6.2 45.1 9 5.5 47.2 9 7.6
47.4 9 1.9 49.9 93.2b 55.9 9 4.0b 52.7 9 0.9b
50.3 92.7b 57.0 9 2.5b 52.9 9 4.5b 55.69 4.5b
a Soapstock (100 g) was refluxed with 100 ml of MEK and phosphoric acid. Following reaction and separation of the MEK phase by centrifugation, the residual was washed twice with MEK, and the combined extracts concentrated to 50 ml. Crystallization was induced by adding glacial acetic acid (1:3 v/v) and allowing the mixture to stand in the dark for 24 h. b Values are not significantly different from the maximum value obtained (PB0.05).
Table 2 Influence of additional methyl ethyl ketone (MEK) extractions of the aqueous residue on the yield, purity, and recovery of gossypol acetic acid isolated from cottonseed soapstocka Number of 50-ml MEK washes
Crude product yield (%)
Purity (%)
0 1 2 3 4 5
1.6090.26 2.059 0.17 2.639 0.11 2.839 0.08 2.699 0.13 2.939 0.07
94.1 9 0.7 90.8 9 4.0 88.0 9 0.3 83.89 1.9 83.7 91.1 83.5 9 0.4
a b c c,d c,d d
Recovery (%) a a,b b c c c
36.7 95.8 45.5 93.5 56.6 92.5 57.9 92.9 54.9 92.2 59.89 1.2
a b c c c c
a Soapstock (100 g) was treated with 100 ml of 1.4 M phosphoric acid in MEK for 2 h. After combining the initial extract with the MEK washings, the MEK phase was concentrated to 50 ml and glacial acetic acid was added in a 1:3 volume ratio. The mixture was left standing for 24 h in the dark. Within a column, values for yield, purity, or recovery with the same letter are not statistically different at PB0.05.
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Table 3 Effect of methyl ethyl ketone (MEK) concentration and acetic acid addition on the yield, purity, and recovery of gossypol acetic acid isolated from cottonseed soapstocka Concentrate volume (ml)
Acetic acid:MEK ratio (v/v) 1:10
1:5
1:3
1:2
60 50 40
Crude product yield (%) 1.429 0.16 2.079 0.39 2.65 90.05b 2.1090.07 2.3290.25 2.71 9 0.09b
2.45 90.10 2.65 9 0.07b 2.89 9 0.18b
2.67 9 0.25b 2.93 9 0.18b 2.89 9 0.04b
60 50 40
Purity (%) 82.793.3 86.79 2.3b 82.491.5
86.4 93.5b 85.6 91.6b 85.1 9 1.1b
86.0 9 1.3b 85.7 9 1.0b 85.4 9 2.1b
87.9 9 1.0b 87.8 9 1.2b 85.5 9 1.4b
60 50 40
Reco6ery (%) 28.89 4.5 44.692.6 46.895.8
43.9 9 9.9 55.4 9 1.8b 56.3 92.0b
51.4 92.6 55.5 9 2.1b 60.1 9 4.0b
57.3 95.1b 62.8 9 3.4b 60.4 91.8b
a Soapstock (100 g) was refluxed with 100 ml of a 1.4 M phosphoric acid MEK solution. Following separation of the MEK phase by centrifugation, the aqueous residual was washed three times with 50 ml of MEK. The MEK–acetic acid solutions were left to stand in the dark for 24 h. b Values are not statistically different from the maximum value obtained (PB0.05).
sidered optimal and were used for the rest of the study. The reduced purity that results because of the additional processing was not detrimental for achieving a final pure product (see following sections).
3.3. Solution concentration and acid addition (Step 3) The initial MEK extract and the three MEK washings were combined and concentrated before adding acetic acid. Because product purity varied only to a small degree over the entire range of conditions studied (82– 88%), product yield and gossypol recovery tracked each other closely. Both yield and recovery were significantly affected by solution concentration and acid addition (P B 0.0001). Purity was also affected by acid concentration (PB 0.03). The highest recovery, 62.8 93.4%, was achieved by concentrating to a final volume of 50 ml (per 100 g of soapstock) and adding 25 ml of acetic acid to give an acetic acid/MEK ratio of 1:2 (Table 3). The increased recovery over the Step 2 results appeared to be due to the increased concentration of acetic acid
in the final crystallization solution. Recoveries of greater than 60% were also achieved by concentrating to 40 ml and adding acetic acid to give a final ratio of either 1:3 or 1:2. Because the rate of MEK stripping decreased progressively as the solution volume was reduced, obtaining a final volume of less than 50 ml (per 100 g of soapstock) was time consuming, and it was not possible to produce concentrates less than 40 ml at the specified rotary evaporator conditions. Because of this difficulty, the optimal conditions carried forward were to concentrate the solution to 50 ml (per 100 g of soapstock) and add 25 ml of acetic acid to give an acetic acid/ MEK ratio of 1:2. At these conditions, the recovered product was 87.89 1.2% gossypol acetic acid.
3.4. Crystallization conditions (Step 4) Since the preliminary experiments indicated that crystallization at 4 or 50°C did not increase gossypol recovery, subsequent work was conducted at room temperature. Most published reports on gossypol recovery use standing
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3.5. Purification of crude product
conditions for crystallization. In this study, such conditions were found to yield reasonable amounts of gossypol, but required long crystallization times (Table 4). Agitation was found to hasten the precipitation and to produce a more consistent product. Without agitation, short ( B 0.5 h) and long (\16 h) crystallization times yielded less pure product (B85% gossypol acetic acid). With agitation, the purity of the recovered samples was less variable ranging between 87.5 and 92.2% for all crystallization times.
To study the influence of additional re-crystallization on product purity, several crude gossypol samples from the optimization process were combined to produce a crude material containing 83.4% gossypol acetic acid. A single re-crystallization of this initial material yielded a 99% gossypol acetic acid product (Table 5). Accounting for both the initial product recovery and the losses associated with the re-crystallization gave a net
Table 4 Effect of crystallization time and agitation on the yield, purity, and recovery of gossypol acetic acid isolated from cottonseed soapstocka Time (h)
0.25 0.5 1 2 4 6 9 16 24 48
Crude product yield (%)
Purity (%)
Without agitation
Without agitation
With agitation
59.5 939.6 a 87.89 6.4 b 91.1 9 1.5 b 91.1 91.6 b 90.69 2.4 b 89.6 91.6 b 88.1 91.2 b 83.4 9 1.8 b 80.09 5.1 a,b 83.89 4.0 b
89.9 9 1.3 89.1 90.6 88.7 92.7 89.4 9 0.7 87.5 9 3.0 89.9 9 4.8 92.0 9 2.1 92.2 90.9 – –
0.88 9 0.66 1.26 90.79 1.64 90.55 1.55 90.67 1.20 9 0.03 2.11 9 0.52 2.00 90.41 2.57 9 0.19 2.88 90.04 2.85 90.17
With agitation a a,b a,b,c a,b,c a,b c,d,e b,c,d d,e e e
2.07 90.23 2.53 9 0.06 2.66 90.08 2.78 9 0.03 2.969 0.08 2.899 0.05 3.0190.03 2.879 0.20 – –
a b b,c c,d d,e d,e e d,e
Recovery (%)
a,b a,b a,b a,b b a,b a a
Without agitation
With agitation
13.9 913.7 a 27.6 917.6 a,b 36.5 912.7 b,c 34.6 9 15.2 b,c 26.5 9 1.0 a,b 46.1 9 10.7 c,d 43.1 9 9.5 b,c,d 52.4 92.9 d 56.3 94.3 d 58.2 90.6 d
45.4 9 4.6 55.2 9 1.6 57.5 90.1 60.8 90.8 63.2 90.4 63.4 9 2.3 67.5 9 0.9 64.6 9 4.3 – –
a b b,c c,d d d,e e d,e
a Extraction conditions: 1.4 M phosphoric acid; 2 h reaction time; three 50-ml washing of the aqueous residue; concentration of the MEK phase to 50 ml/per 100 g initial soapstock and acid addition to the volume ratio of 1:2 acetic acid:MEK. Within a column, values for yield, purity, and recovery with the same letter are not significantly different at PB0.05.
Table 5 Yield, purity, and recovery of crude gossypol acetic acid preparations re-crystallized from methyl ethyl ketone and acetic acida Number of re-crystallizations
Product yieldb (%)
Purity (%)
Re-crystallization recoveryc (%)
Net gossypol recoveryd (%)
Initial materiale 1 2 3
– 76.89 0.4 a 72.290.2 b 68.19 0.2 c
83.4 99.090.6 a 98.79 0.5 a 98.491.3 a
– 91.2 90.5 a 85.5 90.2 b 80.6 90.9 c
– 57.8 90.3 a 54.2 90.2 b 51.19 0.5 c
a
Within a column, values with the same letter are not significantly different at PB0.05. Product yield =(final product weight/weight of initial crude material)×100. c Re-crystallization recovery = (weight of gossypol in re-crystallized sample/weight of gossypol in the initial crude sample)×100. d Net gossypol recovery =(crude product recovery (63.2%)× re-crystallization recovery)/100. e The starting material was the combination of several crude gossypol acetic acid products recovered from soapstock. b
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Table 6 Yield, purity, and recovery of gossypol acetic acid isolated from different cottonseed soapstocks Sample number
Soapstock gossypol concentration (%)
pHa
Yield of crude product (%)
Product purity (%)
Crude product recovery (%)
1 2c 3 4 5
2.5 3.7 5.7 7.6 7.7
10.3 11.1 10.1 9.3 9.6
0.09 9 0.02 3.0 90.1 3.2 90.2 4.5 90.1 5.5 90.2
13.1b 87.5 9 3.0 82.0 91.1 88.7 92.6 88.8 9 1.7
0.4 63.2 9 0.4 40.6 9 2.2 47.2 91.8 56.9 9 1.6
a
pH determined by dispersing 0.50 g of soapstock in 15 ml of deionized filtered water. Due to limited sample, replicates were combined for determination of purity. c Sample used for process development. b
recovery of 57.89 0.3%. Although some increased brightness in the product color was noted with additional re-crystallizations, gossypol recovered by a single re-crystallization assayed the same as gossypol recovered by multiple re-crystallizations (Table 5). Filtering the re-solubilized gossypol during the first recrystallization removed a dark gummy material, which was important for quickly increasing the product purity. The purified product contained carbon and hydrogen in good agreement with expected values (theoretical: 66.4% carbon, 5.9% hydrogen; found: 66.0% carbon, 6.0% hydrogen). Nitrogen was B0.015%. The UV – Vis spectrum in chloroform (peaks: 243, 278, 289, and 366 nm) was identical to the spectrum reported by Boatner (1948). In addition, the spectrum of the 3amino-1-propanol gossypol derivative used for chromatographic detection (peaks: 245, 376, and 400 nm) was identical to the spectrum reported by Hron et al. (1990) for this compound. The melting point of the material was 182.4– 183.5°C in reasonable agreement with values reported by Clark (1927), Campbell et al. (1937) and Murty et al. (1942), which ranged between 180 and 187°C.
3.6. Analysis of different soapstock samples Because of variations in seed quality and processing conditions, soapstock composition is often variable. To determine the influence of this
variability on gossypol recovery, four additional soapstocks with gossypol concentrations ranging between 2.5 and 7.7% were assayed (Table 6). Yield and recovery were very low from the sample containing 2.5% gossypol. Soapstocks with higher gossypol concentrations yielded more gossypol, but little correlation was observed between the gossypol recovery and the initial gossypol concentration of the different samples. Differences in the recoveries did not appear to result from insufficient acid in the hydrolysis step because the sample used for the optimization work had the most residual alkali (highest pH) (Table 6). The limitation appeared to be associated with the crystallization conditions. Pons et al. (1959) suggested that gossypol recovery from gums was limited because of partial solubility of gossypol in the crystallization solution. Because the amount of gossypol lost during crystallization decreases as the purity of the product increases (Pons et al. 1959; Table 5), gossypol solubility in the MEK–acetic acid crystallization liquor appears to be very sensitive to the presence of impurities. Because soapstock tends to be variable in composition, the concentration of co-extracted components in the crystallization solution can also be variable, which may account for the recoveries not correlating with the initial gossypol concentration. Hence, while the conditions described in this report should serve as a reasonable guide for recovering quantities of gossypol from soapstock, maxi-
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mum recovery from any given sample may require some modification of these conditions.
3.7. Comparison with the process from gums Our procedure is similar to the method developed for recovering gossypol from cottonseed gums (Pons et al., 1959). The process described here, however, requires a higher concentration of acid to overcome the residual alkali present in soapstock. In addition, Pons achieved rapid separation of the MEK and aqueous phases on cooling by allowing the reaction mixture to stand. With soapstock, the phases were very slow to separate on standing. Centrifuging the mixture facilitated this process. The crystallization step also occurred more quickly when the isolation was from gums than when the isolation was from soapstock. For gums, a 4-h crystallization period was sufficient to achieve the maximum recovery without mixing. With soapstock, the rate of crystallization was considerably reduced under standing conditions and the time required for maximum recovery was much greater. Agitation of the crystallization liquor, however, increased the precipitation rate sufficiently so that the time required for crystallization was comparable between the two processes. The optimal crude product recovery obtained in this work was 63% from a soapstock sample with an initial gossypol concentration of 3.7%. In comparison, Pons et al., (1959) achieved a recovery of 47– 55% from gums with an initial gossypol concentration of 4.6–5.5%. The improved recovery found in this work from material with a lower concentration of gossypol may be related to differences in the concentration of triglycerides in the gum and soapstock by-products. A number of reports imply that the presence of triglycerides either reduced the rate of gossypol crystallization (Carruth, 1918; Royce et al., 1941) or increased the solubility of gossypol in the crystallization solution (Pons et al., 1959). Miscella refining, although eliminating the production of gums, reduces the amount of oil loss during refining and lowers the concentration of triglycerides in soapstock (Hendrix, 1984). This difference likely accounts for the improved recovery.
4. Conclusions Gossypol in cottonseed soapstock can be recovered in high purity. A crude preparation can be obtained by treating soapstock with acid to release bound gossypol, partitioning the gossypol into an organic phase, and inducing crystallization by adding acetic acid. From a soapstock that initially contained 3.7% gossypol, 63% of the gossypol was recovered as an 87% pure gossypol acetic acid product. A single re-crystallization of this material resulted in a 99% gossypol acetic acid product with an overall recovery of 58%.
Acknowledgements The authors thank Yazoo Valley Oil Co., the National Cottonseed Products Association, and Millard Calhoun for the soapstock samples, Robert Hron, Sr. and Joseph Landry for the AOCS gossypol analyses, and Richard Johnson for help with the statistical analysis.
References American Oil Chemists’ Society, 1998. In: Firestone, D. (Ed.), Official Methods and Recommended Practices of the AOCS, fifth ed. Champaign, IL. Berardi, L.C., Goldblatt, L.A., 1969. Gossypol. In: Liener, I.E. (Ed.), Toxic Constituents of Plant Foodstuffs. Academic Press, New York, pp. 211 – 266. Boatner, C.H., 1948. Pigments in cottonseed. In: Bailey, A.E. (Ed.), Cottonseed and Cottonseed Products. Interscience Publishers, New York, pp. 213 – 363. Campbell, K.N., Morris, R.C., Adams, R., 1937. The structure of gossypol. I. J. Am. Chem. Soc. 59, 1723 – 1728. Carruth, F.E., 1918. Contribution to the chemistry of gossypol, the toxic principle of cottonseed. J. Am. Chem. Soc. 40, 647 – 663. Castillon, L.E., Hall, C.M., Boatner, C.H., 1948. Preparation of gossypol from cottonseed pigment glands. J. Am. Oil Chem. Soc. 25, 233 – 236. Chang, C.J.G., Ghosh, P.K., Hu, Y.F., Brueggemeier, R.W., Lin, Y.C., 1993. Antiproliferative and antimetastatic effects of gossypol on Dunning prostate cell-bearing Copenhagen rats. Res. Commun. Chem. Mol. Pathol. Pharmacol. 79, 293 – 312. Clark, E.P., 1927. Studies on gossypol. I. The preparation, purification, and some of the properties of gossypol, the toxic principle of cottonseed. J. Biol. Chem. 75, 725 – 739.
M.K. Dowd, S.M. Pelitire / Industrial Crops and Products 14 (2001) 113–123 Cosentino, M.J., Matlin, S.A., 1997. Pharmacological developments in male contraception. Exp. Opin. Invest. Drugs 6, 635 – 653. Dowd, M.K., 1996. Compositional characterization of cottonseed soapstocks. J. Am. Oil Chem. Soc. 73, 1287 –1295. Dowd, M.K., Thomas, L.M., Calhoun, M.C., 1999. Crystal and molecular structure of an enantiomeric gossypol-acetic acid clathrate. J. Am. Oil Chem. Soc. 76, 1343 –1350. Flack, M.R., Pyle, R.G., Mullen, N.M., Lorenzo, B., Wu, Y.W., Knazek, R.A., Nisula, B.C., Reidenberg, M.M., 1993. Oral gossypol in the treatment of metastic adrenal cancer. J. Clin. Endocrinol. Metab. 76, 1019 –1024. Hendrix, W.B., 1984. Current practices in continuous cottonseed miscella refining. J. Am. Oil Chem. Soc. 61, 1369 – 1372. Hron, R.J. Sr, Kuk, M.S., Abraham, G., 1990. Determination of free and total gossypol by high performance liquid chromatography. J. Am. Oil Chem. Soc. 67, 182 –187. Hron, R.J. Sr, Kim, H.L., Calhoun, M.C., Fisher, G.S., 1999. Determination of ( + )-, ( − )-, and total gossypol in cottonseed by high-performance liquid chromatography. J. Am. Oil Chem. Soc. 76, 1351 –1355. Kim, H.L., Calhoun, M.C., Stipanovic, R.D., 1996. Accumulation of gossypol enantiomers in ovine tissues. Comp. Biochem. Physiol. 112B, 417 –420. King, W.H., Thurber, F.H., 1953. An improved procedure for the purification of gossypol. J. Am. Oil Chem. Soc. 30, 70– 74. Lin, T.-S., Schinazi, R., Griffith, B.P., August, E.M., Eriksson, B.F.H., Zheng, D.-K., Huang, L., Prussoff, W.H., 1989. Selective inhibition of human immunodeficiency virus type
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1 replication by the ( −) but not the ( +) enantiomer of gossypol. Antimicrob. Agents Chemother., 2149 – 2151. Lindberg, M.C., Naqvi, R.H., Matlin, S.A., Zhou, R.H., Bialy, G., Blye, R.P., 1987. Comparative anti-fertility effects of gossypol enantiomers in male hamsters. Int. J. Androl. 10, 619 – 623. Longmore, J., 1886. Cottonseed oil: its colouring matter and mucilage, and description of a new method of recovering the loss occurring in the refining process. J. Soc. Chem. Ind., 200 – 206. Marchlewski, L., 1899. Gossypol, ein bestandtheil der baumwollsamen. J. Prakt. Chem. 60, 84 – 90. Murty, V.K., Murty, K.S., Seshadri, T.R., 1942. Chemistry of gossypol. Part I. Preparation and properties. Proc. Indian Acad. Sci. A 16, 54 – 61. Pons, W.A. Jr, Pominski, J., King, W.H., Harris, J.A., Hopper, T.H., 1959. Recovery of gossypol from cottonseed gums. J. Am. Oil Chem. Soc. 36, 328 – 332. Qian, S.-Z., Wang, Z.-G., 1984. Gossypol: a potential antifertility agent for males. Annu. Rev. Pharmacol. Toxicol. 24, 329 – 360. Royce, H.D., Harrison, J.R., Hahn, E.R., 1941. Cotton-root bark as a source of gossypol. Oil Soap (Chicago) 18, 27 – 29. SAS Institute Inc., SAS/STAT User’s Guide, Version 6, fourth ed., Vol. 2. Cary, NC, 1989. Tso, W.W., 1984. Gossypol inhibits Ehrlich ascites tumor cell proliferation. Cancer Lett. 24, 257 – 261. Yu, Y.-W., 1987. Probing into the mechanism of action, metabolism and toxicity of gossypol by studying its ( + )and ( − )-stereomers. J. Ethnopharmacol. 20, 65 – 78.
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Recovery of gossypol acetic acid from cottonseed soapstock Michael K. Dowd *, Scott M. Pelitire Commodity Utilization Research Unit, Southern Regional Research Center, ARS, USDA, 1100 Robert E. Lee Bl6d., New Orleans, LA 70124 USA Received 20 September 2000; accepted 13 December 2000
Abstract Gossypol is a yellow pigment found in the cotton plant and is of scientific and medical interest because of its anti-tumor, anti-fertility, and anti-viral properties. In order to support additional studies into the medicinal activity of gossypol, methods are needed to isolate large quantities of the compound in high purity. A process is described to recover gossypol from cottonseed soapstock, a low-value co-product of crude oil refining that can contain concentrations of gossypol as high as 8%. Soapstock is refluxed in acidic methyl ethyl ketone (MEK) to hydrolyze covalently bound gossypol. Upon cooling, the mixture separates into organic and aqueous phases with the gossypol distributing between the two phases. The MEK phase is recovered, and the aqueous phase is re-extracted with additional volumes of MEK. After combining and concentrating the MEK extracts, acetic acid is added to induce gossypol crystallization, which precipitates as an equimolar crystalline complex with acetic acid (gossypol acetic acid). From a soapstock sample containing 3.7% gossypol, 63% of the gossypol was recovered as an 87% gossypol acetic acid product. A single re-crystallization of this crude material yielded a 99% gossypol acetic acid product with an overall recovery of 58%. With different soapstock samples, the yield of crude product was positively correlated with the initial gossypol concentration in the soapstock. Recovery, however, was not well correlated with soapstock gossypol concentration, possibly due to the co-extraction of other components that influence the solubility of gossypol in the crystallization solution. Published by Elsevier Science B.V. Keywords: Cottonseed; Extraction; Foots; Gossypol; Gossypol acetic acid; Soapstock
1. Introduction Gossypol [1,1’,6,6’,7,7’-hexahydroxyl-5,5’-diisopropyl-3,3’-dimethyl-(2,2’-binaphthalene)-8,8’-dicarboxaldehyde] is a polyphenolic yellow compound found in pigment glands distributed * Corresponding author. Tel.: + 1-504-2864339; fax: + 1504-2864367. E-mail address: [email protected] (M.K. Dowd).
throughout the cotton plant (Gossypium sp.). The compound has been associated with a wide range of biological and medicinal activity, including anti-tumor (Tso, 1984; Chang et al., 1993; Flack et al., 1993), anti-fertility (Qian and Wang, 1984; Yu, 1987; Cosentino and Matlin, 1997), and antiviral (Lin et al., 1989) effects. Gossypol is also responsible for toxic effects associated with the overuse of cottonseed products in animal feeds (Berardi and Goldblatt, 1969).
0926-6690/01/$ - see front matter. Published by Elsevier Science B.V. PII: S 0 9 2 6 - 6 6 9 0 ( 0 0 ) 0 0 0 9 4 - 7
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Gossypol exists in a number of tautomeric and isomeric forms. Because of the placement of methyl and hydroxyl groups on the carbon atoms adjacent to gossypol’s binaphthalene bridge (Fig. 1), rotation about this bond is severely restricted. As a consequence, gossypol exhibits atropisomerism and can be found in two enantiomeric forms. The cotton plant produces both forms in a ratio that varies from approximately 60:40 ( + / −) to 45:55 (+/ − ). Several studies indicate that the gossypol isomers contribute disproportionally to the compound’s biological activity, e.g. ( − )-gossypol appears to be responsible for most of the contraceptive effects (Yu, 1987; Lindberg et al., 1987). Because of these differences in activity, there has been interest in developing techniques to separate the (+)- and (− )-gossypol forms. Recently, we reported a crystalline form of gossypol that resolves the enantiomers (Dowd et al., 1999), and we are currently trying to scale up this process to produce the enantiomers in useful amounts. This paper describes a new procedure to prepare the large amounts of gossypol needed to support this work. The earliest isolations of gossypol were from cottonseed soapstock, a by-product of oil refining (Longmore, 1886; Marchlewski, 1899). These initial preparations, however, were quite crude, and the methods were quickly overshadowed by other approaches. Subsequent procedures employed cottonseed or defatted cottonseed (Carruth, 1918), the root bark of the cotton plant (Royce et al., 1941), or isolated pigment glands (Castillon et al., 1948). Pons et al. (1959) proposed using cottonseed gums, a by-product of crude oil refining. At the time, this method was advantageous because gums were highly concentrated in gossypol (4 – 6%) and the use of gums did not interfere with
the utilization of the valuable oil or meal. Unfortunately, the cottonseed industry has subsequently converted from crude oil refining to miscella refining (refining before solvent stripping), and the production of gums has been eliminated. Therefore, an alternative procedure is needed to produce large amounts of research-grade gossypol. Because the concentration of gossypol in cottonseed soapstock can be as high as 8% (Dowd, 1996), a reinvestigation of this material for gossypol recovery is warranted. The general strategy of most recovery methods is to extract gossypol into an organic solvent, concentrate the resulting solution, and precipitate gossypol by adding acetic acid. The product is a crystalline inclusion complex containing an equimolar ratio of gossypol and acetic acid (gossypol acetic acid) (Carruth, 1918). This form of gossypol is relatively stable. It contains the enantiomers in an equimolar ratio, and is widely used as an analytical standard for measuring gossypol in animal tissues and cottonseed products (Hron et al., 1990; Kim et al., 1996; Hron et al., 1999). Methods have been reported to recover pure gossypol from gossypol acetic acid (Carruth, 1918; King and Thurber, 1953; Pons et al., 1959); however, these procedures are tedious and prone to unwanted side reactions. Our process recovers a 99% gossypol acetic acid product from cottonseed soapstock. The process is similar to the method used by Pons et al. (1959) for isolating gossypol acetic acid from cottonseed gums. However, some of the separation steps were modified, and the optimal conditions were different. 2. Material and methods
2.1. Sample preparation and handling
Fig. 1. Structure of gossypol.
Soapstock from a Louisiana miscella-refining cottonseed oil mill was collected immediately downstream of the soapstock centrifuge and cooled rapidly with ice. The National Cottonseed Products Association (Memphis, TN) also provided several additional soapstock samples from different oil mills. All samples were stored at − 20°C until used.
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Fig. 2. Process schematic for the recovery of gossypol from cottonseed soapstock. Process was optimized in four steps (1 – 4) as labeled.
Soapstock gossypol concentration was determined by the AOCS (American Oil Chemists’ Society, 1998) Official Method Ba 8– 78. Alkali concentration was estimated by dispersing 0.5 g of soapstock in 15 ml of deionized filtered water and measuring pH.
2.2. Reco6ery process The procedure (Fig. 2) has five processing operations. These include treating soapstock with acid to hydrolyze bound gossypol, partitioning the gossypol into an organic phase, washing the residual aqueous phase of additional gossypol, concentrating the combined gossypol solution by vacuum distillation, and inducing crystallization by adding acetic acid. For the hydrolysis step, 10091 g of soapstock was weighed into a 1-l flask with 100 ml of methyl ethyl ketone (MEK) containing phosphoric acid and a stir-bar. The flask was connected to a condenser, and the mixture was stirred while heating to reflux. At the end of the hydrolysis step, the flask was cooled to room temperature in an icewater bath, and the contents were transferred to a centrifuge bottle. The mixture was centrifuged at 5000×g for 5 min to separate the emulsion into MEK and aqueous phases. After recovering the
MEK phase, the aqueous phase was contacted with additional 50 ml volumes of MEK. Each MEK/aqueous mixture was briefly shaken by hand and re-centrifuged at 5000× g for 5 min. The combined extracts were filtered through c 4 Whatman paper to remove any residual insoluble material, and a small amount of MEK was used to wash the filter paper. The filtered solution was concentrated with a rotary evaporator using house vacuum ( 25 in Hg vacuum), 5°C condenser water, and 50°C heating water. After transferring the concentrated solution into a graduated cylinder or vial, glacial acetic acid was added to induce crystallization. During the crystallization process, the solution was stored in the dark to prevent photo-oxidation. Crude product was recovered by vacuum filtration through preweighed c 4 Whatman paper, and the gossypol acetic acid precipitate was washed with hexane until the filtrate was colorless. Vacuum was applied until no additional filtrate was collected. The product was stored overnight in a vacuum oven at room temperature to remove residual hexane.
2.3. Process optimization The process was optimized in a series of four experimental studies (Fig. 2) starting with the
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hydrolysis step (Step 1) and working toward the crystallization step (Step 4). Preliminary experiments were conducted to find a reasonable set of starting values for process variables and to bracket the ranges of values needed to ensure that optimal conditions were found. Each step was optimized in sequence carrying over the optimal conditions from the previous steps. Step 1 of the optimization focused on the hydrolysis conditions. Phosphoric acid concentrations of 0.6, 1.0, 1.4, or 1.8 M and reaction times of 0.5, 1.0, 2.0, or 4.0 h were examined. All other steps were kept constant (two 50-ml MEK washings, final concentrate volume of 50 ml per 100 g initial soapstock mass, addition of acetic acid to a 1:3 (v/v) ratio, and crystallization at room temperature for 24 h without agitation). Step 2 tested the effect of including up to five 50-ml MEK washings of the aqueous residue. Step 3 studied concentration conditions (final volume of the MEK –gossypol concentrate and the ratio of MEK-to-acetic acid in the crystallization solution). For this study, the combined gossypol– MEK fractions were concentrated to 40, 50, or 60 ml, and acetic acid was added to give final acetic acid/MEK ratios of 1:10, 1:5, 1:3, or 1:2. Crystallization conditions were studied in Step 4. Time was varied from 0.25 to 48 h with and without agitation. For agitation, a rocker table was used with a 9 20° angle at 90 tilts per minute. Temperature (4°C, room temperature, and 50°C) was also considered in the preliminary experiments used to define starting conditions. To increase the number of crystallization conditions that could be studied, the concentrated gossypol– MEK solutions from four separate 100-g runs were combined and partitioned into 20-ml aliquots.
2.4. Sample purity Purity of crude gossypol products was determined by HPLC against a gossypol acetic acid standard (Kim et al., 1996). Crude product (100 mg) was weighed into a 100-ml volumetric flask and filled to the mark with reagent grade acetonitrile. A 0.5-ml aliquot was transferred to a test tube, mixed with 1.0 ml of a complexing reagent, and heated at 95–100°C for 30 min with the tube
capped. The complexing reagent was 3-amino-1propanol (2 ml), glacial acetic acid (10 ml), and dimethylformamide added to a final volume of 100 ml. After cooling, the derivatized sample was diluted with 9 ml of acetonitrile. The HPLC system consisted of a pumping system and photo-diode array detector (Models 2690 and 996, respectively, Waters Corp., Milford, MA) with Inertsil C18 reverse-phase guard and cartridge columns (SGE, Inc., Austin, TX). The mobile phase was a 0.01 M KH2PO4 buffer adjusted to pH 3 with H3PO4 (20%) in acetonitrile (80%) pumped at a flow rate of 1 ml/min. Injections were 20 ml, and the gossypol complex was detected at 254 nm. A sample of gossypol acetic acid originally isolated and purified from cottonseed gums and believed to be 99% pure was used to prepare a reference standard. This sample was repeatedly re-crystallized by dissolving in acetone (1:6 w/v), filtering with c 4 Whatman paper, and adding one-third volume of acetic acid. The initially dark solutions were shaken until yellow particles were noticeable ( 3 min), and the mixture was stored in the dark for 2 h. Precipitated gossypol acetic acid was isolated by vacuum filtering through c 4 Whatman paper, washing the precipitate with hexane until the filtrate was colorless, and drying under house vacuum overnight at room temperature. The color of the product (bright yellow) improved slightly with each re-crystallization, and the third and final re-crystallized sample was used as the reference material for this work.
2.5. Purification of soapstock gossypol acetic acid The purity of the initial gossypol product was increased by re-crystallization from solutions of MEK and acetic acid. Several samples from the development studies were combined to provide the starting material. For these experiments, 2.090.1 g of crude product was dissolved in MEK (1:7 w/v), filtered through c 4 Whatman paper, and one-half volume of acetic acid was added to induce crystallization. The solutions were agitated in the dark at room temperature for 4 h. Gossypol acetic acid was recovered by filtering through c4 Whatman paper, rinsing with
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hexane until the filtrate was colorless, and vacuum drying overnight at room temperature. The process was repeated up to three times. The initial filtration step was omitted after the first re-crystallization. The purity of the re-crystallized products was determined following the procedure outlined for the crude samples. Testing of the final gossypol acetic acid product was conducted to ensure authenticity. The melting point was determined with a capillary melting apparatus, and the carbon, hydrogen, and nitrogen concentrations were determined by combustion methods. The UV– Vis spectrum was recorded in chloroform with a spectrophotometer (Model UV160 U, Shimadzu Scientific Instruments, Inc., Columbia, MD), and the spectrum of the di-3-amino-1-propanol gossypol complex was determined with the HPLC photo-diode detector.
2.6. Calculations and statistics Crude product yield was calculated as the weight of precipitated product divided by the initial wet weight of soapstock. Purity was measured as the detector area per unit mass of the sample divided by the detector area per unit mass of the standard. Gossypol recovery was calculated as the amount of gossypol in the crude product divided by the amount of gossypol in the soapstock. For the purification study, similar definitions were used except that the yield and recovery values were referenced to the starting crude gossypol product. In addition, a net gossypol recovery was defined to account for the recovery from the initial isolation process and the recovery from purification. All reported values were expressed on a percent basis. Experiments were conducted in triplicate. The SAS GLM analysis of variance procedure (SAS Institute Inc., 1989) was used to test for main and interaction effects on yield, purity, and recovery. In addition, pairwise ‘t’-tests were computed with the MEANS statement (h = 0.05). When two variables were included in the experimental design (Steps 1 and 3), least square means for the interaction effects were also tested.
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3. Results and discussion
3.1. Hydrolysis conditions (Step 1) Acid concentration, reaction time, and the interaction of acid concentration and time all significantly affected crude product yield (PB 0.001) (Table 1). The yield was very low at 0.6 M phosphoric acid, but increased at the higher concentrations. With sufficient acid concentration and hydrolysis time, product yield appeared to reach a ‘plateau’ that represented the maximum yield. Acid concentration also affected product purity (PB0.0001) (Table 1). At low acid concentration (0.6 M), purity varied from 39 to 64% over the reaction times studied. At greater acid concentrations (]1.0 M), product purity was significantly higher (82–87%). Both the low yield and low purity contributed to the low gossypol recovery (B 10%) achieved at low acid concentration. Hydrolysis time was important at the 1.0 M phosphoric acid concentration, but less important at higher acid concentrations (1.4 and 1.8 M). As for product yield, a ‘plateau’ state was reached with sufficient acid and reaction time, which corresponded to the maximum recovery. The strong influence of acid concentration on product yield and recovery confirms that soapstock gossypol is mostly in a bound form (Dowd, 1996). The highest gossypol recovery was 57.09 2.5% with 1.8 M phosphoric acid and a 1-h reaction time. This recovery, however, was not significantly different from the recoveries obtained with 1.8 M acid concentration and other reaction times or the recoveries obtained with a 1.4 M acid concentration and reaction times greater than 1.0 h. To reduce acid use, the optimal reaction conditions were taken to be 1.4 M phosphoric acid and a 2-h hydrolysis period.
3.2. Extraction of aqueous residue (Step 2) Additional MEK washing of the aqueous residual affected the crude product yield, purity, and recovery (P B0.0001). Yield significantly increased with the first three extractions, but did not
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ditional washings beyond the third did not significantly increase gossypol recovery (Table 2). To achieve the highest gossypol purity, only the initial extraction should be conducted. Recovery of gossypol, however, improves with up to three additional washings of the aqueous residue. In this work, our first concern was to achieve high recovery. Therefore, three extractions were con-
increase with the fourth or fifth extraction (Table 2). Product purity decreased with the first three additional washings (Table 2). As with yield, recovery increased with additional washing, but the increases were less pronounced because of the decreasing trend associated with product purity. Three washings increased the recovery 58% over the recovery obtained without the washings. Ad-
Table 1 Effect of hydrolysis conditions on the yield, purity, and recovery of gossypol acetic acid isolated from cottonseed soapstocka Reaction time (h)
Phosphoric acid concentration (M) 0.6
1.0
1.4
1.8
0.5 1.0 2.0 4.0
Crude product yield (%) 0.38 9 0.25 0.299 0.09 0.289 0.21 0.3690.26
1.13 9 0.30 2.00 90.15 2.14 9 0.19 2.24 9 0.26
2.22 9 0.14 2.39 90.08 2.69 9 0.07b 2.51 9 0.08b
2.48 90.13b 2.69 90.26b 2.63 90.11b 2.76 90.20b
0.5 1.0 2.0 4.0
Purity (%) 49.09 36.3 64.8 9 12.6b 39.89 32.0 48.19 36.0
84.9 91.5b 85.8 9 7.1b 86.4 9 3.4b 86.0 94.7b
87.4 92.3b 85.5 9 6.7b 84.9 94.7b 86.2 92.3b
83.2 9 4.2b 87.1 9 5.2b 82.5 96.4b 82.6 9 3.1b
0.5 1.0 2.0 4.0
Reco6ery (%) 6.09 5.1 4.79 2.3 3.7 94.5 5.595.8
23.4 96.1 42.0 9 6.2 45.1 9 5.5 47.2 9 7.6
47.4 9 1.9 49.9 93.2b 55.9 9 4.0b 52.7 9 0.9b
50.3 92.7b 57.0 9 2.5b 52.9 9 4.5b 55.69 4.5b
a Soapstock (100 g) was refluxed with 100 ml of MEK and phosphoric acid. Following reaction and separation of the MEK phase by centrifugation, the residual was washed twice with MEK, and the combined extracts concentrated to 50 ml. Crystallization was induced by adding glacial acetic acid (1:3 v/v) and allowing the mixture to stand in the dark for 24 h. b Values are not significantly different from the maximum value obtained (PB0.05).
Table 2 Influence of additional methyl ethyl ketone (MEK) extractions of the aqueous residue on the yield, purity, and recovery of gossypol acetic acid isolated from cottonseed soapstocka Number of 50-ml MEK washes
Crude product yield (%)
Purity (%)
0 1 2 3 4 5
1.6090.26 2.059 0.17 2.639 0.11 2.839 0.08 2.699 0.13 2.939 0.07
94.1 9 0.7 90.8 9 4.0 88.0 9 0.3 83.89 1.9 83.7 91.1 83.5 9 0.4
a b c c,d c,d d
Recovery (%) a a,b b c c c
36.7 95.8 45.5 93.5 56.6 92.5 57.9 92.9 54.9 92.2 59.89 1.2
a b c c c c
a Soapstock (100 g) was treated with 100 ml of 1.4 M phosphoric acid in MEK for 2 h. After combining the initial extract with the MEK washings, the MEK phase was concentrated to 50 ml and glacial acetic acid was added in a 1:3 volume ratio. The mixture was left standing for 24 h in the dark. Within a column, values for yield, purity, or recovery with the same letter are not statistically different at PB0.05.
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Table 3 Effect of methyl ethyl ketone (MEK) concentration and acetic acid addition on the yield, purity, and recovery of gossypol acetic acid isolated from cottonseed soapstocka Concentrate volume (ml)
Acetic acid:MEK ratio (v/v) 1:10
1:5
1:3
1:2
60 50 40
Crude product yield (%) 1.429 0.16 2.079 0.39 2.65 90.05b 2.1090.07 2.3290.25 2.71 9 0.09b
2.45 90.10 2.65 9 0.07b 2.89 9 0.18b
2.67 9 0.25b 2.93 9 0.18b 2.89 9 0.04b
60 50 40
Purity (%) 82.793.3 86.79 2.3b 82.491.5
86.4 93.5b 85.6 91.6b 85.1 9 1.1b
86.0 9 1.3b 85.7 9 1.0b 85.4 9 2.1b
87.9 9 1.0b 87.8 9 1.2b 85.5 9 1.4b
60 50 40
Reco6ery (%) 28.89 4.5 44.692.6 46.895.8
43.9 9 9.9 55.4 9 1.8b 56.3 92.0b
51.4 92.6 55.5 9 2.1b 60.1 9 4.0b
57.3 95.1b 62.8 9 3.4b 60.4 91.8b
a Soapstock (100 g) was refluxed with 100 ml of a 1.4 M phosphoric acid MEK solution. Following separation of the MEK phase by centrifugation, the aqueous residual was washed three times with 50 ml of MEK. The MEK–acetic acid solutions were left to stand in the dark for 24 h. b Values are not statistically different from the maximum value obtained (PB0.05).
sidered optimal and were used for the rest of the study. The reduced purity that results because of the additional processing was not detrimental for achieving a final pure product (see following sections).
3.3. Solution concentration and acid addition (Step 3) The initial MEK extract and the three MEK washings were combined and concentrated before adding acetic acid. Because product purity varied only to a small degree over the entire range of conditions studied (82– 88%), product yield and gossypol recovery tracked each other closely. Both yield and recovery were significantly affected by solution concentration and acid addition (P B 0.0001). Purity was also affected by acid concentration (PB 0.03). The highest recovery, 62.8 93.4%, was achieved by concentrating to a final volume of 50 ml (per 100 g of soapstock) and adding 25 ml of acetic acid to give an acetic acid/MEK ratio of 1:2 (Table 3). The increased recovery over the Step 2 results appeared to be due to the increased concentration of acetic acid
in the final crystallization solution. Recoveries of greater than 60% were also achieved by concentrating to 40 ml and adding acetic acid to give a final ratio of either 1:3 or 1:2. Because the rate of MEK stripping decreased progressively as the solution volume was reduced, obtaining a final volume of less than 50 ml (per 100 g of soapstock) was time consuming, and it was not possible to produce concentrates less than 40 ml at the specified rotary evaporator conditions. Because of this difficulty, the optimal conditions carried forward were to concentrate the solution to 50 ml (per 100 g of soapstock) and add 25 ml of acetic acid to give an acetic acid/ MEK ratio of 1:2. At these conditions, the recovered product was 87.89 1.2% gossypol acetic acid.
3.4. Crystallization conditions (Step 4) Since the preliminary experiments indicated that crystallization at 4 or 50°C did not increase gossypol recovery, subsequent work was conducted at room temperature. Most published reports on gossypol recovery use standing
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3.5. Purification of crude product
conditions for crystallization. In this study, such conditions were found to yield reasonable amounts of gossypol, but required long crystallization times (Table 4). Agitation was found to hasten the precipitation and to produce a more consistent product. Without agitation, short ( B 0.5 h) and long (\16 h) crystallization times yielded less pure product (B85% gossypol acetic acid). With agitation, the purity of the recovered samples was less variable ranging between 87.5 and 92.2% for all crystallization times.
To study the influence of additional re-crystallization on product purity, several crude gossypol samples from the optimization process were combined to produce a crude material containing 83.4% gossypol acetic acid. A single re-crystallization of this initial material yielded a 99% gossypol acetic acid product (Table 5). Accounting for both the initial product recovery and the losses associated with the re-crystallization gave a net
Table 4 Effect of crystallization time and agitation on the yield, purity, and recovery of gossypol acetic acid isolated from cottonseed soapstocka Time (h)
0.25 0.5 1 2 4 6 9 16 24 48
Crude product yield (%)
Purity (%)
Without agitation
Without agitation
With agitation
59.5 939.6 a 87.89 6.4 b 91.1 9 1.5 b 91.1 91.6 b 90.69 2.4 b 89.6 91.6 b 88.1 91.2 b 83.4 9 1.8 b 80.09 5.1 a,b 83.89 4.0 b
89.9 9 1.3 89.1 90.6 88.7 92.7 89.4 9 0.7 87.5 9 3.0 89.9 9 4.8 92.0 9 2.1 92.2 90.9 – –
0.88 9 0.66 1.26 90.79 1.64 90.55 1.55 90.67 1.20 9 0.03 2.11 9 0.52 2.00 90.41 2.57 9 0.19 2.88 90.04 2.85 90.17
With agitation a a,b a,b,c a,b,c a,b c,d,e b,c,d d,e e e
2.07 90.23 2.53 9 0.06 2.66 90.08 2.78 9 0.03 2.969 0.08 2.899 0.05 3.0190.03 2.879 0.20 – –
a b b,c c,d d,e d,e e d,e
Recovery (%)
a,b a,b a,b a,b b a,b a a
Without agitation
With agitation
13.9 913.7 a 27.6 917.6 a,b 36.5 912.7 b,c 34.6 9 15.2 b,c 26.5 9 1.0 a,b 46.1 9 10.7 c,d 43.1 9 9.5 b,c,d 52.4 92.9 d 56.3 94.3 d 58.2 90.6 d
45.4 9 4.6 55.2 9 1.6 57.5 90.1 60.8 90.8 63.2 90.4 63.4 9 2.3 67.5 9 0.9 64.6 9 4.3 – –
a b b,c c,d d d,e e d,e
a Extraction conditions: 1.4 M phosphoric acid; 2 h reaction time; three 50-ml washing of the aqueous residue; concentration of the MEK phase to 50 ml/per 100 g initial soapstock and acid addition to the volume ratio of 1:2 acetic acid:MEK. Within a column, values for yield, purity, and recovery with the same letter are not significantly different at PB0.05.
Table 5 Yield, purity, and recovery of crude gossypol acetic acid preparations re-crystallized from methyl ethyl ketone and acetic acida Number of re-crystallizations
Product yieldb (%)
Purity (%)
Re-crystallization recoveryc (%)
Net gossypol recoveryd (%)
Initial materiale 1 2 3
– 76.89 0.4 a 72.290.2 b 68.19 0.2 c
83.4 99.090.6 a 98.79 0.5 a 98.491.3 a
– 91.2 90.5 a 85.5 90.2 b 80.6 90.9 c
– 57.8 90.3 a 54.2 90.2 b 51.19 0.5 c
a
Within a column, values with the same letter are not significantly different at PB0.05. Product yield =(final product weight/weight of initial crude material)×100. c Re-crystallization recovery = (weight of gossypol in re-crystallized sample/weight of gossypol in the initial crude sample)×100. d Net gossypol recovery =(crude product recovery (63.2%)× re-crystallization recovery)/100. e The starting material was the combination of several crude gossypol acetic acid products recovered from soapstock. b
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Table 6 Yield, purity, and recovery of gossypol acetic acid isolated from different cottonseed soapstocks Sample number
Soapstock gossypol concentration (%)
pHa
Yield of crude product (%)
Product purity (%)
Crude product recovery (%)
1 2c 3 4 5
2.5 3.7 5.7 7.6 7.7
10.3 11.1 10.1 9.3 9.6
0.09 9 0.02 3.0 90.1 3.2 90.2 4.5 90.1 5.5 90.2
13.1b 87.5 9 3.0 82.0 91.1 88.7 92.6 88.8 9 1.7
0.4 63.2 9 0.4 40.6 9 2.2 47.2 91.8 56.9 9 1.6
a
pH determined by dispersing 0.50 g of soapstock in 15 ml of deionized filtered water. Due to limited sample, replicates were combined for determination of purity. c Sample used for process development. b
recovery of 57.89 0.3%. Although some increased brightness in the product color was noted with additional re-crystallizations, gossypol recovered by a single re-crystallization assayed the same as gossypol recovered by multiple re-crystallizations (Table 5). Filtering the re-solubilized gossypol during the first recrystallization removed a dark gummy material, which was important for quickly increasing the product purity. The purified product contained carbon and hydrogen in good agreement with expected values (theoretical: 66.4% carbon, 5.9% hydrogen; found: 66.0% carbon, 6.0% hydrogen). Nitrogen was B0.015%. The UV – Vis spectrum in chloroform (peaks: 243, 278, 289, and 366 nm) was identical to the spectrum reported by Boatner (1948). In addition, the spectrum of the 3amino-1-propanol gossypol derivative used for chromatographic detection (peaks: 245, 376, and 400 nm) was identical to the spectrum reported by Hron et al. (1990) for this compound. The melting point of the material was 182.4– 183.5°C in reasonable agreement with values reported by Clark (1927), Campbell et al. (1937) and Murty et al. (1942), which ranged between 180 and 187°C.
3.6. Analysis of different soapstock samples Because of variations in seed quality and processing conditions, soapstock composition is often variable. To determine the influence of this
variability on gossypol recovery, four additional soapstocks with gossypol concentrations ranging between 2.5 and 7.7% were assayed (Table 6). Yield and recovery were very low from the sample containing 2.5% gossypol. Soapstocks with higher gossypol concentrations yielded more gossypol, but little correlation was observed between the gossypol recovery and the initial gossypol concentration of the different samples. Differences in the recoveries did not appear to result from insufficient acid in the hydrolysis step because the sample used for the optimization work had the most residual alkali (highest pH) (Table 6). The limitation appeared to be associated with the crystallization conditions. Pons et al. (1959) suggested that gossypol recovery from gums was limited because of partial solubility of gossypol in the crystallization solution. Because the amount of gossypol lost during crystallization decreases as the purity of the product increases (Pons et al. 1959; Table 5), gossypol solubility in the MEK–acetic acid crystallization liquor appears to be very sensitive to the presence of impurities. Because soapstock tends to be variable in composition, the concentration of co-extracted components in the crystallization solution can also be variable, which may account for the recoveries not correlating with the initial gossypol concentration. Hence, while the conditions described in this report should serve as a reasonable guide for recovering quantities of gossypol from soapstock, maxi-
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mum recovery from any given sample may require some modification of these conditions.
3.7. Comparison with the process from gums Our procedure is similar to the method developed for recovering gossypol from cottonseed gums (Pons et al., 1959). The process described here, however, requires a higher concentration of acid to overcome the residual alkali present in soapstock. In addition, Pons achieved rapid separation of the MEK and aqueous phases on cooling by allowing the reaction mixture to stand. With soapstock, the phases were very slow to separate on standing. Centrifuging the mixture facilitated this process. The crystallization step also occurred more quickly when the isolation was from gums than when the isolation was from soapstock. For gums, a 4-h crystallization period was sufficient to achieve the maximum recovery without mixing. With soapstock, the rate of crystallization was considerably reduced under standing conditions and the time required for maximum recovery was much greater. Agitation of the crystallization liquor, however, increased the precipitation rate sufficiently so that the time required for crystallization was comparable between the two processes. The optimal crude product recovery obtained in this work was 63% from a soapstock sample with an initial gossypol concentration of 3.7%. In comparison, Pons et al., (1959) achieved a recovery of 47– 55% from gums with an initial gossypol concentration of 4.6–5.5%. The improved recovery found in this work from material with a lower concentration of gossypol may be related to differences in the concentration of triglycerides in the gum and soapstock by-products. A number of reports imply that the presence of triglycerides either reduced the rate of gossypol crystallization (Carruth, 1918; Royce et al., 1941) or increased the solubility of gossypol in the crystallization solution (Pons et al., 1959). Miscella refining, although eliminating the production of gums, reduces the amount of oil loss during refining and lowers the concentration of triglycerides in soapstock (Hendrix, 1984). This difference likely accounts for the improved recovery.
4. Conclusions Gossypol in cottonseed soapstock can be recovered in high purity. A crude preparation can be obtained by treating soapstock with acid to release bound gossypol, partitioning the gossypol into an organic phase, and inducing crystallization by adding acetic acid. From a soapstock that initially contained 3.7% gossypol, 63% of the gossypol was recovered as an 87% pure gossypol acetic acid product. A single re-crystallization of this material resulted in a 99% gossypol acetic acid product with an overall recovery of 58%.
Acknowledgements The authors thank Yazoo Valley Oil Co., the National Cottonseed Products Association, and Millard Calhoun for the soapstock samples, Robert Hron, Sr. and Joseph Landry for the AOCS gossypol analyses, and Richard Johnson for help with the statistical analysis.
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