POLY-UNSATURATED FATTY ACID ESTER COMPOSITION AND ITS PREPARATION
The present invention relates to a therapeutic preparation comprising 30 to 100 gamma- [...] acid (6, 9, 12-octadecatrienoic) in the form of alkyl ester and 0 The invention also relates to a composition of polyunsaturated fatty acids useful in the therapeutic preparation above. It is known that gamma-linolenic acid is an important intermediate in a metabolic process pu- comprising in a living body from which [...] linoleic acid, oads and terminating one essential fatty acid/acid- [...] [...] (5, 8, 11, 14-eicosatetraenoic acid). In the metabolic process linoleic acid-gamma-linolenic acid acid [...] -gamma-nic [...] s " arachidonic acid, only a portion of the linoleic acid administered to the living organism is converted into arachidonic acid. Instead, substantially all of the acid [...] administered to a living body is rapidly converted into arachidonic acid [J. E. Kea. d, D. E. Button, J. [...]. Chem. 229,575 (1957), J. Biol. Chen. 2 ^, 3385 (1960)], while the [...] -linolenic acid (9, 12, 15-octadecatrienoic acid), does not contribute to the metabolic process. As a result. of those facts that the gamma acid. -linolenic fatty acid is a biologically very important. On the other hand, the abnormal fat metabolism [...] various diseases was investigated from many years but it is not possible, although it has been safely elucidated. In general, in diseases such as liver diseases, diabetes, atherosclerosis, ln formation of the fatty acids in the liver or blood is abnormal in most cases. Therefore, it is observed that the proportion of the linoleic acid or arachidonic acid, which are essential fatty acids, that decreases and. that of. [...] pe increases. In such a case, is administered in general a linoleate. Uh such treatment improves the rate of linoleic acid but no improvement ' the level of arachidonic acid. It is considered that the cause is the abnormality metabolism :, linoleic acid arachidonic acid. In the metabolism of linoleic acid into arachidonic rat normal, as has been described above, only a portion of the linoleic acid is converted into arachidonic acid through the gamma-linolenic acid, but most of the gamma-linolenic acid is metabolized is arachidonic acid. The Requester has been found that in the living [...], ls converting [...] gamma-linolenic acid is inhibited and therefore that administration of linoleic acid or an ester or salt thereof of individuals suffering diseases aforementioned-s does not result in enhanced formation of the arachidonic acid in the body. the Requester has been found further that the administration of a therapeutic composition comprising 30 to 100 However, there is no natural oil containing [...] reasonably large range-linolenic acid. On the other hand, its chemical synthesis is industrially difficult. The seed oil of plants, such as those of Oe- [...][...].Symphytum officinale, etc, contains about 10 to 25 has' [...] -linolenic acid. Cos oils But also contain a saturated fatty acid so a excessively large amount of these oils to obtain the. amount of '- [...] -linolenic set as required. In general, a natural substance containing gama-linolenic acid also contains a large amount of " [...] linoleic having physico-chemical properties similar to those of the gamma-linolenic acid so it is difficult to obtain a gamma-linolenic acid having a high concentration by a convenient method and conventional fractionation of fatty acids. Therefore, the methods mentioned in J. Chem. coulter. Japan, 62,1083 (1942), [...]" Chem. Acta, [...], 1017 and (1958) J. Chem. coulter. 2779,5270 (1901) are not substantially applicable to the selective separation of gamma-linolenic acid to large scale. the Requester has been found that the application of a special method using a urea adduct or a silver complex to d '. obtain a composition containing 30 to 100 gamma-linolenic acid and 0 to 70 linoleic acid from an oil containing gamma-linolenic acid. The method c-is described below in detail. Can be used in the practice of methods es any obtained from a natural substance containing gamma- [...]. For example, can be used a fatty acid containing gamma-linolenic acid or an ester thereof [...] (methyl or. ethyl) obtained by any method known and an oil from plant seeds or known as Oenothera [...]Symphytum officinale or microbial or protozoa. In the method forming the urea adduct, the raw material (fatty acids or esters) is dissolved in a suitable solvent as an alcohol the toi and the urea is added to the solution. 0r = agitates a mixture and heated, is then cooled causing it to precipitate the fatty acid or its c- [...] as adducts with urea and the [...] -saturated fatty acids or their esters remain in the solvent without forming do such adducts. Precipitates is separated by filtration and the filtrate is concentrated. Is added distilled water and a small amount of HCl to the concentrate and depletes the mixture by means of ether. The ether extract through mth distillation to obtain the fatty acid composition poly-unsaturated according to the invention. The [...] - [...] amount of acid or its ester in fatty acid composition obtained is all the greater, the amount of urea to the raw material is relatively high. The will generally use 5 to 5 parts by weight of urea per part of raw material. It is preferable to use, as a solvent, methanol or ethanol in a proportion of four to six times the raw material. When, the cooling temperature to precipitate the adducts as said is less than 0 °C, the purity of the gamma-linolenic acid is reduced. Therefore, the temperature should be 0° Is known to e. [...] a non-saturated fatty acid or its ester form a complex. The Requester has been found that there was a difference between the equilibrium constants of a complex of gamma-linolenic acid and a complex of silver and silver linoleic acid and. the. Requester utilising this phenomenon in a chromatographic column to obtain the. acid composition poly-unsaturated according to the invention. Therefore, silver is held in a column of a exchange resin acid, a cation exchange resin or silica gel and a solution of the raw material fed to the column to adsorb the fatty acids (or their esters) in the form of complexes of silver in the column. The column is then subjected to 1' elution using a suitable solvent. Specifically is the first elution fatty acids esters or unsaturated lower and then that of the more saturated fatty acids or esters; and is collected a fraction of gamma-linolenic acid or its ester. Examples of specific for the elution solvents are alcohols, aqueous alcohols, ethers and petroleum ethers. Los thus collected fractions are concentrated to recover the desired composition. The method of forming the urea adduct or silver complex ester gives a composition of a. ^ [...][...] fatty non-saturated s whose contents are: [...] ester of gamma-linolenic acid: [...] 0 to 30, preferably 90 to 100 linoleic acid ester [...]: 0 to 70,0, preferably 0 to 10 the oil composition is a colorless or light yellow in colour and has the following properties boiling point, acid value saponification index iodine value stability 170-180 °C/1-2 not more than 2 mm 185-192 200-270 to unstable air, stable under nitrogen. As therapeutic agent the above composition can be administered as such had can be used in forms conventionally used in the pharmaceutical preparations such as capsules, emulsions, powders, preparations for injections or feed supplement, andc. the following examples illustrate the present invention in greater detail by reference to the graphical sc appended 1 and 2 showing the effects that the present composition on the liver. Example 1 Is dissolved in 20 liters of methanol 4 kg of a mixture of fatty acid esters [...] (acid composition of 6,3 0 palmitic acid, 2.3 'h stearic acid, > oleic acid 11.0 f, / 72.3' _AOE296A0AO> linoleic acid and 7.6 0 gamma-lino [...] ) obtained by ester exchange seedoil to ' Oenothera [...]. To this solution is added 20 kg urea. The moves air present in the reactor by the [...] and the mixture is heated at reflux for ten minutes stirring. Then the mixture is cooled to 20° 0 ; it is forms a precipitate. The precipitate is separated by filtration c-t the filtrate is concentrated under reduced pressure in a nitrogen stream. A. tire rim to residue is distilled water and a small amount of a dilute solution of hydrochloric acid, and tu depletes means of ether. Q & filtered off crystalline precipitate formed and subjected to the same operation as above by adding 20 litres of methanol saturated with urea. After two or three do such operations, there is essentially no acidmethyl ester 1 [...][...] - [...] in the urea adduct. The ether extract the fatty acid composition to analysis by gas liquid chromatography and back is collected fractions higher amounts of [...] -linolenic acid that the usual concentration (75° Example 2 Is dissolved in 40 litres of ethanol 8 kg of ethyl esters of a mixture of fatty acids (6.3 Example 5 Is treated by means of an excess of silver nitrate solution 1.5 liter of ion-exchange resin "Dowex 50 WX 8" (type H). The resin is placed in a column and washed first with distilled water, then ethanol. Then is dissolved 200 g of a composition ethyl esters of fatty acids (35,5 composition? gamma-linolenic acid and 65 [...] 4 Is treated 200 g of silica gel with an aqueous solution of silver nitrate and is heated to activate. The _AOE280A2AO> silica gel is then mixed with 100 g of "celite" and is arranged on a column. Is dissolved 30 g of the mixture of fatty acid esters mentioned in the example 3 in a small amount of petroleum ether. The solution in the column and then it is subjected to the elution using petroleum ether containing Example 5 Is treated in the same manner as in the [...] two hundred grams of a mixture of fatty acids obtained by hydrolysis using a base of the same fatty acid ester that in the example 3. Each of the fractions is distilled of the methanol eluate to flush. The depletes the residue with petroleum ether. The extract is analyzed by gas liquid chromatography and are combined fractions having a high content of gamma-linolenic acid; is obtained. 20,8 g [...] -linolenic acid having a purity of 100 Example 6 Are administered 200 or 400 mg/kg of [...] -linolenate ethyl or 400 ng/kg of ethyl linoleate once per day to six weeks from rats subjected to a fatty [...] régine free. Sacrifice year The rats. May of two, five, ten and fifteen days and examining the formation of fatty acid in the liver phospholipids. [...] the counter in Table I the. rate of production of the arachidonic [...], the administration of 200 mg/kg gamma- [...] time provides at higher levels than product that the administration of 400 m. Linoleate g/kg. Table i Coloration arachidonic acid in the liver phospholipids Probe [...] -linolenate ethyl k 200 mg/ [...] -linolenate ethyl 400 mg/kg ethyl linoleate 400 ng/kg 10 15 6.68 8.41 8.11 7.06 +0.11+0.99+0, + 27 0.84 12.00 16.70 20.00 20.85+0.43 +1.22 +1.02+0, 83,13.52 22.71 27.47 28.28 +0.70 +0.65 +0,35 +1.40 10.56 13.01 18.35 16.88+0.27 example 7+0.62 ±1.02 +1.17. Is administered once per day for 200 hundred days or 500 mg/kg of a mixture of fatty acid esters [...] (composition 33 > ' acid [...] - [...] and 67 rl linoleic acid) and pure [...] Linoleate to rats subjected to a regime free fat is searched and the composition of the fatty acids in the liver phospholipids. The results exposed in Table II demonstrate production of arachidonic acid in the phospholipids is higher in the group receiving 200 mg/kg of [...] -linolenate that in the group receiving 500 ng/kg Linoleate. Coloration arachidonic acid in the -phospholipids liver ^ Composition: 33 67 EXAMPLE g Is administered once per day for four weeks 200 mg/kg of gamma-linolenate methyl methyl or Linoleate and between time 0.1 ml/kg of carbon tetrachloride for two weeks to cause liver [...]. the comparison with the control group shows that the greensickness. us of the group having received gamma-linolenate have improved serum alkaline phosphatase and recovery of arachidonic acid in the liver phospholipids. Table iii Arachidonic acid and alkaline phosphatase in the treated rat by carbon tetrachloride [...] Probe Gamma linolenate of [...][...] -. 200 mg/kg [...] methyl 200 mg/kg Phosphatase 43,3 ± 24.2 113,1 ± 31.7 55.0+35.81 63,7 24.1± Arachidonic acid fi 22.50+1.95 20.87+2.92 * Units Xing-Armstrong Example 9 The speed for one week at a free fat young rats age four weeks. Their then administered The 2^the weight of the food of gamma-linolenate ethyl; or ethyl linoleate. The sacrifice is at the end of three days and testing for the composition of fatty acids in the liver and the serum triglyceride. Figures 1 and 2 shows results obtained. [In both figures: A = large free; > B = [...] ethyl | C = gamma- [...] ethyl]. L-. figure 1 shows the effect of the acid supply gran on the content of fatty acids in the liver rat (ordered: ug fatty acid/g liver) [...] acids [...] -tri Moreover 1' increase of the triglyceride, which is a ^ [...][...], in the [...] is considered from the fact that the fatty substance neutral present in the liver is released into the blood as [...] -lipoprotein. It has been found that, in such an action, there are more that do [...] - [...][...]. Example 10 Is administered a proportion of six capsules by a preparation days do [...] - [...] (containing 50 Table iv Percent of the fatty acids composition back serum phospholipids 1,240,513. Fatty acid esters. ONO PHARMACEUTICAL CO. Ltd. 13 Sept., 1968, No. 43790/68. Heading C2C. [Also in Division A5] Mixtures of at least 30% methyl or ethyl esters of γ-linolenic acid and not more than 70% methyl or ethyl esters of linoleic acid are prepared by heating a solution of a raw material containing γ-linolenic acid or its methyl or ethyl ester with urea, filtering and recovering the ester composition from the filtrate or alternatively by passing through a column of silver adsorbed on a carrier, eluting with a solvent and collecting the enriched eluate. The raw materials may be mixtures of fatty acids containing γ-linolenic acid or the methyl and ethyl esters or oils from plant seeds, e.g. Oenothera homarkiana or Symphytum officinale or from microbes or protozoa. The compositions are suitable for the treatment of diseases accompanied by abnormal liver metabolism. The. comparison do the fatty acid composition of prior and after administration of the gamma-linolenate shows a decrease (p-" CO, 01) palmitic acid C-| g. ^ and an increase (p < C. 0,05) ^20*4 arachidonic acid* Example 11 Is administered; orally to ten patients suffering from atherosclerosis (304 mg/dl on average blood cholesterol) for six months, six capsules containing 400 mg per capsule 35 i 't of gamma-lino the score and 65 ethyl After a months of administration, the average content of blood into cholesterol is fallen to 236 ng/dl and at the end of six months to 227 mg/dl. [...] m e A therapeutic composition for the treatment of diseases associated with abnormal lipid metabolism, comprising 30 to 100 [...] ester (methyl or ethyl) of 1 ' [...] - [...] acid and 0 to 70 Group
Time (days)
Group Dose Arachidonic acid Probe 3,93 ± 0.76 Ester [...] fatty acid ** 23.40+2.90 methyl linoleate 200 ng/kg 18,05 1.89± Fatty acid methyl ester* 26.56+2.20 methyl linoleate 500 mg/kg 17.21+3.06 * alkaline
in a patient suffering from liver cirrhosis
C 14:0 G is; o 16*1° C 1s; 0 8:1 ° LEVEL I C 18:2 ° 20:4 Before 0,5 32,7 2,0 14,0 14,0 17,8 2,1 8,0 After the administration of revived- [...] 0,6 30,7 1,9 13,5 13,2 17,4 3,3 9,2