7KLVPDWHULDOPD\EHSURWHFWHGE\&RS\ULJKWODZ7LWOH86&RGH
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`Table I. Proximate Composition of Krill Samples
`(as Percentage of Total)
`frozen
`krill
`77.7 —
`14.0
`3.01.
`2.74
`97.5
`
`moisture
`protein*
`fat
`ash
`total
`
`freeze-dried
`krill
`7.83
`52.0
`16.7
`12.2
`88.7
`
`‘
`
`krill
`mean
`1.75
`60.7
`11.5
`13.4
`87.4
`
`- #Total nitrogen was multiplied by 6.25.
`
`Table II. Yields of Oils Extracted from Krill Samples with
`Supercritical Carbon Dioxide
`
`sample
`freeze-dried
`krill
`
`krill meal
`
`19.7
`
`16.2
`
`amt, g/100 g sample
`rec,°
` extr
`resid
`total
`extractn
`%
`oil
`lipid’?
`_lipid?®
`condn*®
`95
`11.2
`7.6
`250/40
`98
`11.7:
`7.9
`250/60
`99
`115
`81
`250/80
`84
`4.5
`8.1
`250/40
`81
`4.1
`9.0
`250/60
`17
`4.0
`84
`250/80
`80
`4.0
`9.0
`400/40
`*Pressure, kg/cm?/temperature, °C.
`’Measured by the Bligh—
`Dyer method.
`‘Recovery = [extr oil + resid lipid]/total lipid x
`100.
`:
`
`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% DEGSon 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-CO,-extracted
`oils and in theresidual lipids was analyzed by the method
`reported by Yamaguchiet al. (1983).
`RESULTS AND DISCUSSION
`Table I shows the proximate compositionsof the krill
`samples. Total recoveries of components of 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 kriil
`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
`lowrecoveries in the freeze-dried krill and the krill meal
`may be also accounted for by chitin, contents of which
`should be higher than that in the frozen krill. |
`Figure 1 presents the SC-CO, extraction curves with
`time of oils from the freeze-dried krill and krill meal with
`SC-CO, at 250 kg/cm? and 80 °C. For freeze-dried krill;
`extraction of oil practically terminated in 3-4 h andafter
`the use of 2-3 kg of CO, 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 CO,. Reproducibility in the
`experiments was very high.
`Table II shows the yields of oils extracted with SC-CO,
`underdifferent conditions. In every sample the extracted
`oil was fluid and bright red from carotenoids. When the
`temperature was increased from 40 to 60 and 80 °C ata
`fixed pressure of 250 kg/cm? and when the pressure was
`increased from 250 to 400 kg/cm?at a fixed temperature
`of 40 °C,yields ofextracted oils remained almost constant.
`
`
`
`gC-CO, Extraction of Oils from Antarctic Krill
`
`J. Agric. Food Chem., Vol. 34, No. 5, 1986
`
`905
`
`10
`
`
`
`
`
`yieldsofoils(g/100gsample)
`
`Freeze-dried
`krill
`
`Krill meal
`
`715
`
`
`
`4
`
`2
`
`3
`
`4
`
`5
`
`6
`
`(kg)
`co, flux
`Figure 1. Extraction curves of krill oils with supercritical carbon
`dioxide at 250 kg/cm? and 80 °C.
`
`:
`
`In this connection, Brogle (1982) reported that the solvent
`power of SC-CO, for organic substances is highly de-
`pendent on its pressure and temperature; at low pressures,
`around 100 kg/cm?, solvent power drops with rising tem-
`peratures, and at pressures higher than approximately 150
`kg/cm?, 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/cm? and 40 °C), nearly all oils ex-
`tractable with SC-CO, were recovered. Further, yields of
`krill meal oil were one-thirdof those of freeze-dried krill
`oil. The lower yields from meal oil are probably attrib-
`utable to the fact that theoil of the krill meal was in part
`_ deteriorated by oxidation or polymerization to such an
`extent that only limited extraction occurred with SC-CO..
`Judging from this result, we think that SC-CO, extraction
`is suitable methodto obtain undenaturedoils from meals
`of marine origin.
`To determine the composition of the extractedoils, we
`partitioned them bychromatography ona 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 theoils extracted with SC-CO, were composed ex-
`clusively of nonpolar lipids with practically no polar lipids.
`Figure 2 shows TLCpatterns of the oils extracted with
`SC-CO,and the residual lipids. By co-TLC with standard
`reagents, the main componentof the extracted oils was
`found tobe 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
`nonpolarlipid (spot 10) was found to be present in the oil
`from the krill meal, but it remained unidentified. The free
`fatty acid content of theoil from the krill meal was lower
`than that of the freeze-dried krill. Because of their rapid
`deterioration, some of free fatty acids in the meal should
`have been denatured during manufacture and storage of
`the meal.
`As shown in chromatogram II, trace amounts of some
`nonpolarlipids (spots 3-9) were observed, but almostall
`the residual lipids (spot 11) were not affected when de-
`AKER EXHIBIT 2002 Page 2
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`
`LITERATURE CITED
`Association of Official Analytical Chemists, Inc. Official Methods
`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
`
`Yamaguchi et al,
`
`Table IV. Content and Composition of Carotenoids in Oils
`Extracted from Freezée-Dried Krill with Supercritical.
`Carbon. Dioxide
`
`carotenoid
`total content, mg/100 g oil
`composition, %
`astaxanthin diester
`astaxanthin monoester
`astaxanthin
`unidentified
`*Préssure: 250 kg/em?.
`composition.
`
`temp of extractn,? °C
`40
`60
`.
`43.5-50.4
`19.1-24.38
`:
`
`:
`
`80
`7.0-8.7
`b
`78-83
`48-58
`b
`13-15
`33-44
`b
`3-4
`5-7
`b
`ol
`0-2
`’Unable to be determined due to de
`
`
`
` I
`
`fréeze-dried krill at a fixed pressure of 250 kg/cm? and
`stepwise increase of temperatures.
`In previous papers
`‘(Yamaguchi‘etal., 1983; Miki et al., 1983), we reported that
`the carotenoids of the Antarctic krill consist almost ex-
`clusively of astaxanthin andits 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-CO,. Although Zosel (1978) pointed out
`that SC-CO,is suitable for the isolation of thermally labile ©
`substancesbecause of its low critical temperature, the
`above finding indicates that some compoundslikeasta-
`xanthin could be unstable under high pressures of SC-S-
`CO, at a temperature, for example 80 °C, that never in-
`duces the decomposition of astaxanthin under atmospheric
`‘Table Ill. Fatty Acid Composition of Oils Extracted from
`pressure (Mikiet al., 1983). Attention should be paid to
`Krill Samples with Supercritical Carben Dioxide*
`this fact in the extraction of natural products with SC-CO).
`freeze-
`freeze-
`In spite of such a disadvantage, we proved that SC-CO,
`.
`dried
`dried
`- is effective in obtaining nonpolar lipids from Antarctic krill
`
`fatty acid-krill krillmeal fatty acid krill krill meal
`
`
`
`by a simple, one-step extraction that excludes the phos-
`12:0
`0.25
`0.28
`18:4
`1.98
`3.21
`pholipids that have hampered theutilization ofkrilloils.
`14:0
`17.42
`19.08
`20:1
`1.82
`1.86
`15:0
`0.28
`0.21
`20:3
`0.15
`0.09
`Jn this connection, several workers (Stahl et al., 1980;
`16:0 ~
`22.05
`18.78
`20:4wW6 +
`1.08
`0.55
`Friedrich and List, 1982) have reported the removal of
`16:1
`11.78
`16.30
`22:1
`polar lipids from seed oils with SC-CO,, but the reason why
`17:1
`1.09
`1.70
`20:403
`polar lipids can be removed with SC-CO,has not yet been
`18:0
`1.47
`1.33
`20:5
`elucidated.
`18:1
`21.37
`22.40
`22:5
`As shown in Table III, the oils extracted from krill
`18:2
`2.24
`3.56
`22:6
`18:3
`0.30
`0.26
`24:1
`samples with SC-CO, contained fairly high proportions of
`unidenti-
`eicosapentaenoic acid (EPA), which is known as a miedi-
`fied
`cally useful substance (Needleman et al., 1979). Recently,
`Krukonis (1984) reported that, by using SC-CO,forthe
`fractionation of fish oils, EPA could be concentrated to
`15% from8%. Used in this manner, SC-CO, extraction
`will produce useful substances from aquatic organisms.
`Abbreviations Used: SC-CO,, 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 of
`Nippon Suisan Kaisha, Ltd., for their valuable advice.
`Thanksaré also due to N. Ando and K. Kajiyama, Iwatani
`& Co., Ltd., for their help in SC-CO, extraction experi
`ments. We also thank Professor George J. Flick, De-
`partment of Food Science and Technology, Virginia Po-
`lytechnic Institute and State University, for his review of
`this paper.
`Registry No. EPA, 1553-41-9; CO:, 124-38-9; astaxanthin,
`472-61-7.
`
`IZ’
`TEL
`Figure 2. Thin-layer chromatogramsof krill oils extracted with
`supercritical carbon dioxide (I) and the residual lipids (II and IID:
`A, freeze-dried krill; B, krill meal. Stationary phase: silica gel
`60F954 in chromatograms I-III. Mobile phase: petroleum eth-
`er-diethyl ether—acetic acid (90:10:1) in chromatograms Land IL
`chloroform—methanol-water (65:25:4)
`in chromatogram III.
`Identification: 50% H,SO, in chromatograms Tand I]; Dragen-
`dorff reagent in chromatogram III.
`
`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/cm, 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 Ill, when the residual lipids were developed
`using chloroform-methanol-water (65:25:4) andvisualized
`with Dragendorff reagent, at least three orange-red spots
`(12-14) of phospholipids were detected. These results
`confirmed that the SC-CO, 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-CO, at a pressure of 250 kg/cm? and
`a temperature of 60 °C. Essentially no differences were
`noticed in the fatty acid compositionsof the oils extracted
`underother 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-CO, from. the
`
`’ v
`
`
`
`
`
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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..London 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, 8. 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. 8. The Principles of Gas Extraction; Mills
`and Boon, Ltd.: London, England, 1971.
`Stahl, E.; Schutz, 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 December9, 1985. Revised manuscriptre-
`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
`andtissue extracts by a simple procedure. Bovine plasma (0.11.0 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 removedinterfering substances. The bottom column (transfer pipet) contained 0.3-1.0
`g of wet anion-exchangeresin in the phosphate form and trapped the estrogens through their phenolic
`hydroxyl group. The estrogens were then eluted with acetic acid in acetonefollowing 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.
`
`
`acetone extractn
`0.1
`0.5
`‘1.0
`1.0
`ether extractn
`1.0
`
`2
`4
`8
`8°
`
`4
`
`;
`
`1.0 (8)4
`1.0 (8)
`1.5 (5)
`1.5 (5)
`
`1.0 (3)
`
`1.0 (2)°
`2.0 (3)
`8.0 (4)
`- 3.0 (4)
`,
`1.0 (2)
`
`INTRODUCTION
`Table I. Column Conditions for Isolation of [>H]Estradiol
`Added to Bovine Plasma and Tissue Extracts Using the
`Partial purification of estrogens extracted from animal
`Alumina Jon-Exchange Columns
`tissues and fluids is necessary prior to most methodsof
`resin
`extr appl
`quantitation. The methods currently employedin routine
`to col,
`basic alumina,
`suspensn,
`analysis of estrogens such as paper chromatography (Shutt,
`
`sample mL mL g
`
`
`1969), Sephadex LH-20 (Sjovall and Nystrom,.1968;
`Murphy, 1970; Mikhail et al., 1970; Murphy and Diez
`A. Bovine Plasma (mL)
`D’Aux, 1975), and Celite column cleanup (Korenmanet
`al., 1969; Abraham et al., 1970) are tedious with reported
`recoveries of only 65-85%. Aqueoussolutions of estrogens
`had also been purified by ion exchange (Jarvenpaaetal.,
`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
`
`:
`
`B. Bovine Tissues (1 g/8 mL of Acetone)
`2.0 (3)
`2
`1.0 (3)
`liver
`2.0 (8)
`2
`1.0 (3)
`muscle
`2.0 (8)
`2
`1.0 (3)
`heart
`2.0 (3)
`2
`1.0 (8)
`kidney ©
`“Total volume 95% acetone (mL) to wash alumina column.
`>Total volume 10% HOAc in acetone (mL)to elute [*H]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 CheAikeESkc&tXHIBIT 2002 Page 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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