7KLVPDWHULDOPD\EHSURWHFWHGE\&RS\ULJKWODZ7LWOH86&RGH
`
`AKER EXHIBIT 2002 Page 1
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`

`

`30302 Extraction of Oils from Antarctic Krill
`
`J. Agric. Food Chem, Vol. 34, No. 5, 1986
`
`905
`
`Table I. Proximate Composition of Krill Samples
`/_———_.—'_—‘
`(as Percentage of Total)
`frozen
`freeze-dried
`kr1ll
`krill
`krill
`mean
`77.7 '
`7.83
`1.75
`14.0
`52.0
`60.7
`3.01
`16.7
`11.5
`2.74
`12.2
`13.4
`97.5
`88.7
`87.4
`
`moisture
`protein“
`fat
`ash
`total
`
`.
`
`L
`
`' “Total nitrogen was multiplied by 6.25.
`
`Table II. Yields of Oils Extracted from Krill Samples With
`supercritical Carbon Dioxide
`
`10
`
`
`
`
`
`Yieldsofoils(gr/100,9sample)
`
`-15
`
`
`
`Freeze-dried
`krill
`
`Krill meal
`
`Figure 1. Extraction curves of krill oils with supercritical carbon
`dioxide at 250 kg/cm2 and 80 °C.
`
`CO2 flux
`
`(kg)
`
`.
`
`In this connection, Brogle (1982) reported that the solvent
`power of SC—002 for organic substances is highly de—
`pendent on its pressure and temperature; at low pressures,
`around 100 kg/cmz, solvent power drops with rising tem—
`peratures, and at pressures higher than approximately 150
`kg/ cm2, its solvent power increases. For the krill samples,
`however, the amounts of extracted oils were almost con—
`stant regardless of pressures and temperatures examined.
`That is, even under the lowest temperature/pressure
`combination (250 kg/cm2 and 40 °C), nearly all oils ex—
`tractable With SC-C02 were recovered. Further, yields of
`krill meal oil were one--third of those of freeze—dried krill
`oil. The lower yields from meal oil are probably attrib—
`utable to the fact that the oil of the krill meal was in part
`. deteriorated by [oxidatiOn or polymerization to such an
`extent that only limited extraction occurred with SC-CO2.
`Judging from this result, we think that SC-002 extraction
`is suitable method to obtain undenatured oils from meals
`of marine origin.
`To determine the composition of the extracted oils, We
`partitioned them by chromatography on a silica gel column
`using a mobile phase of chloroform and methanol. Ap-
`proximately 100% of the oils from both krill samples was
`recovered in the fraction eluted with chloroform, proving
`that the oils extracted With SCéCOZ were composed ex.-
`' clusively of nonpolar lipids with practically no polar lipids.
`Figure 2 shows TLC patterns of the oils extracted with
`80-C02 and the residual lipids. By co--TLC with standard
`reagents, the main component of the extracted oils was
`found to be triglycerides (spot 3) and the minor compo-
`nents were identified as hydrocarbon (1), cholesteryl ester
`(2), free fatty acids (4), diglycerides (5), cholesterol (7),
`monoglycerides (8), and carotenoids (6 and 9), as illustrated
`in chromatogram I.
`In addition, a small amount of a
`nonpolar lipid (spot 10) was found to be present1n the oil
`from the krill meal, but it remained unidentified The free
`fatty acid content of the oil from the krill meal was lower
`than that of the freeze—dried krill. Because of their rapid
`deterioration, some 0f free fatty acidsIn the meal should
`have been denatured during manufacture and storageof
`the meal.
`
`As shown in chromatogram II, trace amounts of some
`nonpolar lipids'(spots 3—9) were observed, but almost all
`the residual lipids (spot 11) were not affected when de-
`
`AKER EXHIBIT 2002 Page 2
`
`
`
`
`amt, g/ 100 g sample
`rec,”
`extr
`resid
`total
`extractn
`
` sample condn" oil lipidb lipidb %
`
`
`
`
`freeze-dried
`250/ 40
`11.2
`7.6
`95
`krill
`250/60
`11.7‘
`7.9
`98
`250/80
`11.5
`8.1
`99
`250/ 40
`4.5
`8.1
`84
`250/60
`4.1
`9.0
`81
`250/80
`4.0
`8.4
`77
`400/40
`4.0
`9.0
`80
`
`krill meal
`
`19.7
`
`16.2
`
`”Pressure, kg/ cmz/temperature, °C. bMeasured by the Bligh—
`Dyer method. cRecovery = [extr oil + resid lipid] /total lipid X
`100.
`V
`
`After saponification and then esterification with boron
`trifluoride—methanol complex, the fatty acid composition
`of the oils was determined by GLC With a Shimadzu GC-
`5A gas chromatograph using a glass column (2 m X 3 mm)
`packed with 10% DEGS on Celite 545 (80—100 mesh) at
`a temperature of 200 °C, with nitrogen carrier gas at a flow
`rate of 25 mL/min. The temperature of the detector and
`injection port was 250 °C.
`The composition of carotenoids in the SC-Cog-extracted
`oils and in the residual lipids was analyzed by the method
`reported by Yamaguchi et a1. (1983).
`RESULTS AND DISCUSSION
`
`Table I shows the proximate compositions of the krill
`samples. Total recoveries of components 0f the freeze-
`dried krill and the krill meal were rather low, because of
`the application of the Soxhlet method with diethyl ether
`for the extraction of lipid. The lipid of krill which contains.
`high proportions of polyunsaturated fatty acids and
`phospholipids (Mori and Hikichi, 1976), deteriorates rap—
`idly and diethyl ether insoluble lipid increases when krill
`is dehydrated or treated at high temperatures.
`In this
`connection, the lipid content of the krill meal was found
`to be 16. 2% when measured by the method of Bligh and
`Dyer (1959) with chloroform—methanol. Furthermore, the
`low recoveries in the freeze-dried krill and the krill meal
`may be also accounted for by chitin, contents of Which
`should be higher than thatin the frozen krill. _
`Figure 1 presents the SC-COZ extraction curves with
`time of oils from the freeze-dried krill and krill meal With
`SC--C02 at 250 kg/ cm2and 80 °C. For freeze-dried krill,
`extraction of oil practically terminated1n 3—4 h and after
`the use of 2—3. kg of C02 at a flow rate of about 0.6 kg/ h
`Extraction of the krill meal oil, hoWever, ceased after 2—3
`h and the use of 1—2 kg of C02. Reproducibility in the
`Experiments was very high.
`Table II shows the yields of oils extracted with SC—CO2
`under different conditions. In every sample the eXt'racted
`Oil was fluid and bright red from carotenoids. When the
`temperature was increased from 40 to 60 and 80 °C at a
`fixed pressure of 250 kg/ cm and when the pressure was
`Increased from 250 to 400 kg/cm2 at a fixed temperature
`0f 40 °C, yields of extracted oils remained almost constant.
`
`
`
`AKER EXHIBIT 2002 Page 2
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`

`

`
`
`
`
`Yamaguchi et aL
`
`Table IV. Content and Composition of Carotenoids in Oils
`Extracted from Freeze-Dried Krill with Supercritical.
`Carbon Dioxide '
`
`
`temp of extractn,“ °C
`
`40
`60
`.
`carotenoid
`80
`
`total content, mg/ 100 g oil
`composition, %
`.
`astaxanthin diester
`astaxanthin monoester
`astaxanthin
`unidentified
`
`~
`
`43.5—50.4
`.
`48—58
`33—44
`5—7
`0—2
`
`19.1—24.3
`.
`
`78—83
`13—15
`3—4
`1
`
`7.0—8.7
`b
`
`5
`b
`b
`
`“Pressure: 250 kg/cm2. bUnable to be determined due to de-
`composition.
`
`freeze-dried krill at a fixed pressure of 250 kg/cm2 and
`stepwise increase of temperatures.
`In previous papers
`' (Yamaguchi’et al., 1983; Miki et al., 1983), we reported that
`the carotenoids of the Antarctic krill consist almost ex-
`clusively of astaxanthin and its esters and that their sta-
`bilities against heat and organic solvents are in the order
`of astaxanthin diester, astaxanthin monoester, and. asta—
`xanthin. It is evident that the astaxanthin tends to be
`, decomposed according to their instabilities during ex-
`traction with SC-COZ. Although Zosel (1978) pointed out
`that SC-C02 is suitable for the isolation of thermally labile ‘
`substances because of its low critical temperature, the
`above finding indicates that some compounds like asta-
`xanthin could be unstable under high pressures of SOS-
`CO2 at a temperature, for example 80 °C, that never in-
`duces the decomposition of astaxanthin under atmospheric
`pressure (Miki et al., 1983). Attention should be paid to
`this fact in the extraction of natural products with 80—002.
`In spite of such a disadvantage, we proved that SC-C02
`. is effective in obtaining nonpolar lipids from Antarctic krill
`by a simple, one-step extraction that excludes the phos-
`pholipids that have hampered the utilization of krill oils.
`In this connection, several workers (Stahl et al., 1980;
`Friedrich and List, 1982) have reported the removal of
`polar lipids from seed oils with SC-COZ, but the reason why
`polar lipids can be removed with 80—002 has not yet been
`elucidated.
`As shown in Table III, the oils extracted from krill
`samples with SC—002 contained fairly high proportions of
`.eicosapentaenoic acid (EPA), which is known as a medi-
`cally useful substance (Needleman et al., 1979). Recently,
`Krukonis (1984) reported that, by using SC—C02 for the
`fractionation of fish oils, EPA could be concentrated to
`15% from, 8%. Used in this manner, SC-C02 extraction
`will produce useful substances from aquatic organisms.
`Abbreviations Used: SC-COZ, supercritical carbon
`dioxide; EPA, eicosapentaenoic acid.
`ACKNOWLEDGMENT
`
`We are indebted to Dr. Y. Miyake, Director of the
`Central Research Institute of Iwatani & Co., Ltd., and Dr-
`T. Fujita, DirectOr of the Central Research Laboratory 0f
`Nippon Suisan Kaisha, Ltd., for their valuable advice;
`Thanks are also due to N. Ando and K. Kajiyama, Iwatal?1
`& Co., Ltd., for their help in SC-002 extraction expen—
`ments. We also thank Professor George J. Flick, De-
`partment of Food Science and Technology, Virginia P0-
`1ytechnic Institute and State University, for his review Of
`this paper.
`Registry No. EPA, 1553-41—9; COZ, 124-38-9; astaxanthin:
`472—61—7.
`
`LITERATURE CITED
`
`Association of Official Analytical Chemists, Inc. Official Method5
`of Analysis of the Association of Official Analytical Chemists:
`14th ed.; AOAC: Washington, DC, 1984.
`
`AKER EXHIBIT 2002 Page 3 ,
`
`906
`
`J. Agric. Food Chem, Vol. 34, No. 5, 1986 ‘
`A
`B
`A
`B
`
`A
`
`B
`
`
`
`II‘
`
`III
`
` I
`
`Figure 2. Thin-layer chromatograms of krill oils extracted with
`‘supercritical carbon dioxide (1) and the residual lipids (II and III):
`A, freeze-dried krill; B, krill meal. Stationary phase: silica gel
`60F254 in chromatograms I—III. Mobile phase: petroleum eth—
`er—diethyl ether—acetic acid (90:10:1) in chromatograms I and II;
`chloroform—methanol—Water (65:25:4)
`in chromatogram III.
`Identification: 50% H2804 in chromatograrns I and II; Dragen—
`dorff reagent in chromatogram III.
`«
`.
`
`'Table III. Fatty Acid Composition of Oils Extracted from
`Krill Samples with Supercritical Carbon Dioxide“
`freeze-
`freeze-
`dried
`'
`dried
`
`fatty acid
`krill
`krill meal
`fatty acid
`krill
`krill meal
`12:0
`0.25
`0.28
`18:4
`1.98
`3.21
`14:0
`17.42
`19.08
`20:1
`1.82
`1.86
`15:0
`0.28
`0.21
`20:3
`0.15
`0.09
`16:0 '
`22.05
`18.78
`20:4w6 +
`1.08
`0.55
`16:1
`11.78
`16.30
`22:1
`17:1
`1.09
`1.70
`20:4w3
`18:0
`1.47
`1.33
`20:5
`18:1
`21.37
`22.40
`22:5
`18:2
`2.24
`3.56
`2226
`18:3
`0.30
`0.26
`24:1
`unidenti-
`fied
`
`0.32
`. 11.36
`0.20
`4.44
`0.32
`0.08
`
`0.30
`6.62
`0.18
`2.71
`0.42
`0.16
`
`“Conditions of extraction: 250 kg/cmz, 60 °C.
`
`eloped with petroleum ether—diethyl ether—acetic acid
`(90:10:1), indicating that the residual lipids were composed
`almost exclusively of polar lipids together with denatured
`and polymerized lipids. Furthermore, as shown in chro-
`matogram III, when the residual lipids were developed
`using chloroform—methanol—water (65:25z4) and visualized
`with Dragendorff reagent, at least three orange-red spots
`(12—14) of phospholipids were detected. These results
`confirmed that the SC—002 extraction of krill samples
`yielded only nonpolar undenatured oils, as mentioned
`above.
`.
`Table III shows the fatty acid compositions of the oils
`extracted with SC-COZ at a pressure of 250 kg/ cm2 and
`a temperature of 60 °C. Essentially no differences were
`noticed in the fatty acid compositions of the oils extracted
`under other conditions. Whereas the main fatty acids were
`14:0, 16:0, 16:1, 18:1, and 20:5 in oils of both freeze—dried
`krill and krill meal, significant differences were found
`between krill meal and freeze-dried krill oils in the 16:1,
`20:5, and 22:6 acids.
`Table IV shows the content and composition of caro—
`tenoids in the oils extracted with SC-COZ from. the
`
`» v
`
`
`
`
`
`AKER EXHIBIT 2002 Page 3
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`

`

`
`
`
`
`J. Agric. Food Chem. 1986,, 34, 907—910
`
`‘
`
`907
`
`Bligh, E. G.; Dyer, W. J. Can. J. Biochem. Physiol. 1959, 37, 911.
`Brannolte, H. D.; Mangold, H. K; Stahl, E. Chem. Phys. Lipids
`1983, 33, 297.
`Brogle, H. Chem. Ind. 1982, 19, 385.
`Friedrich, J. P.; List, G. R. J. Agric. Food Chem. 1982, 30, 192.
`Hannay, J. B.; Hogarth, J. Proc. R. Soc..Lond0n 1879, 29, 324.
`Hubert, P.; Vitzthum, O. G. Angew. Chem, Int. Ed. Engl. 1978,
`17, 710.
`Kaufmann, W.; Biernoth, G.; Frade, E.; Merk, W.; Precht, D.;
`Timmen, H. 'Milchwissenschaft 1982, 37, 92.
`Krukonis, V. JAOCS 1984, 61, 698.
`Miki, W.; Toriu, N.; Kondo, Y.; Murakami, M.; Yamaguchi, K.;
`Konosu, S.; Satake, M.; Fujita, T. Bull. Jpn. Soc. Fish. 1983,
`49, 1417.
`Mori, M.; Hikichi, S. Rep. Cent. Res. Lab. Nippon Suisan Co.
`1976, No. 11,12.
`
`Needleman, P.; Raz, A.; Mikes, S. M.; Ferrendelli, J. A.; Sprecher,
`H. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 944.
`Paul, P. F.; Wise, W. S. The Principles of Gas Extraction;,Mills
`and Boon, Ltd.: London, England, 1971.
`Stahl, E.; S'chutz, E.; Manglod, H. K. J. Agric. Food Chem. 1980,
`28, 1153.
`Wilke, G. Angew. Chem, Int. Ed. Engl. 1978, 17, 701.
`Yamaguchi, K.; Miki, W.; Toriu, N.; Kondo, Y.; Murakami, M.;
`Konosu, S.; Satake, M.; Fujita, T. Bull. Jpn. Soc. Sci. Fish.
`1983, 49, 1411.
`.
`Zosel, K. Ger. Pat. 2005 243, 1974.
`Zosel, K. Angew. Chem, Int. Ed. Engl. 1978, 17, 701.
`
`Received for review December 9, 1985. Revised manuscript re-
`ceived April 2, 1986. Accepted April 22, 1986.
`
`Isolation of Estrogens in Bovine Plasma and Tissue Extracts Using -
`Alumina and Ion-Exchange Microcolumns
`‘
`
`
`
`Marjorie B. Medina* and Daniel P. Schwartz
`
`Estrogens (estradiol, estrone) in picogram quantities can be isolated quantitatively from bovine plasma
`and tissue extracts by a simple procedure. Bovine plasma (0.1—1.0 mL) was extracted with either acetone
`or ether while tissues (1 g) were extracted with acetone. Extracts were passed through two disposable
`plastic tubes vertically arranged in tandem. The top column (5-mL pipet tip) contained 1-1.5 g of dry
`basic alumina and removed interfering substances. The bottom column (transfer pipet) contained 0.3—1.0
`g of wet anion-exchange resin in the phosphate form and trapped the estrogens through their phenolic
`hydroxyl group. The estrogens were then eluted with acetic acid in acetone following a thorough washing
`of the columns. Recoveries greater than 95% were obtained when extracts of bovine plasma and tissue
`extracts of liver, kidney, and heart were spiked’with either tritiated 176-estradiol or estrone. This
`and accuracy over traditional methods employed
`technique offers the advantages of simplicity, rapidity,
`routinely in the purification of estrogens.
`
`
`INTRODUCTION
`
`Partial purification of estrogens extracted from animal
`tissues and fluids is necessary prior to most methods of
`quantitation. The methods currently employed in routine
`analysis of estrogens such as paper chromatography (Shutt,
`1969), Sephadex LH—20 (Sjovall and Nystrom, 1968;
`Murphy, 1970; Mikhail et al., 1970; Murphy and Diez
`D’Aux, 1975), and Celite column cleanup (Korenman'et
`al., 1969; Abraham et al., 1970) are tedious with reported
`recoveries of only 65—85%. Aqueous solutions of estrogens
`had also been purified by ion exchange (Jarvenpaa et al.,
`1979) with reported recoveries of 50—90%. Covey and
`co—workers'(1984) also used an anion-exchange resin for
`purification of diethylstilbestrol and dienestrol.
`This paper describes a relatively simple and rapid
`technique that can quantitatively isolate the estrogens
`(estradiol, estrone) from acetone extracts of bovine blood
`plasma and tissues " for subsequent chromatographic
`analysis. This study is a preliminary report on the de-
`velopment of screening methods to detect and measure
`residues of estrogens in the blood and edible tissues of
`food—producing animals given growth-promoting hormones ‘
`
`
`Table I. Column Conditions for Isolation of [3H]Estradiol
`Added to Bovine Plasma and Tissue Extracts Using the
`Alumina Ion-Exchange Columns
`extr appl
`resin
`to col,
`suspensn,
`basic alumina,
`mL sample mL g
`
`
`
`
`
`acetone extractn
`0.1
`0.5
`1.0
`1.0
`ether extractn
`1.0
`
`A. Bovine Plasma (mL)
`
`2
`4
`8
`8c
`
`4
`
`1.0 (3)“
`1.0 (3)
`1.5 (5)
`1.5 (5)
`
`1.0 (3)
`
`,
`
`1.0 (2)5
`2.0 (3)
`3.0 (4)
`- 3.0 (4)
`'
`1.0 (2)
`
`-
`
`B. Bovine Tissues (1 g/ 8 mL of Acetone)
`2.0 (3)
`2
`1.0 (3)
`liver
`2.0 (3)
`2
`1.0 (3)
`muscle
`2.0 (3)
`2
`1.0 (3)
`heart
`2.0 (3)
`2
`1.0 (3)
`kidney .
`“Total volume 95% acetone (mL) to wash alumina column.
`bTotal volume 10% HOAc in acetone (mL) to elute [3H]estradiol.
`° Plasma/ acetone mixture added directly to column with glass wool
`on top of alumina bed.
`
`Eastern Regional Research Center, Agricultural Re—'
`search Service, U.S. Department of Agriculture, Phila—
`delphia, Pennsylvania 19118.
`
`This article not subject to U.S. Copyright. Published 1986 by the American CheA'LQEdRQEXHlBlTZOOZ Pa e 4
`
`such as 176-estradiol and to ascertain their absence such
`that safe and wholesome food can be delivered to con-
`sumers.
`
`AKER EXHIBIT 2002 Page 4
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