the pharmacologically effective ingredients to be used in the present invention are α , β - unsaturated higher fatty acids represented by the following formula : ( wherein n represents 10 , 12 , 14 or 16 ) or pharmaceutically acceptable salts thereof specifically , 2 - tetradecenoic acid , 2 - hexadecenoic acid , 2 - octadecenoic acid , and 2 - eicosenoic acid and pharmaceutically acceptable salts thereof . the pharmaceutically acceptable salts include alkali metal salts such as the sodium or potassium salts . these α , β - unsaturated fatty acids have been found to possess the effect of curing diabetes and the effect of improving lipid metabolism . the present invention provides a medicinal composition comprising the above - described α , β - unsaturated fatty acid or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier , which can be used for prophylaxis and remedy of hyperlipemia , arteriosclerosis , liver troubles such as fatty liver and hepatitis , and for prophylaxis and remedy of diabetes . further , the present invention provides a method for treating diabetes or improving lipid metabolism which comprises administering to a human being a therapeutically effective dosage of a composition comprising a compound of the following formula : ( wherein n represents 10 , 12 , 14 or 16 ) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier . the dosage of the effective ingredients of the present invention varies depending upon the degree of disease , weight of a human to be treated therewith , etc . but , in the case of oral administration to adults , they are administered 2 to 3 times a day , with each administration being conducted generally in a dosage of 50 to 500 mg , preferably 100 to 300 mg . as the preparation form for administering the effective ingredients , there are tablets , capsules ( including ordinary capsules retaining granular medicines and soft capsules retaining a liquid sealed therein ), and liquids for oral administration . also , they may be used in the form of , for example , injections or drops to be introduced into the body . the compounds of the present invention are so susceptible to solvolysis or oxidation that they , in any preparation form , are preferably kept out of contact with ambient atmosphere . water and alcohol are not generally suitable solvents because they accelerate hydrolysis of the effective ingredients . thus , as a liquid carrier , anhydrous and oily substances such as oleic acid , linoleic acid , linolenic acid , etc . are preferably used . as a specific example of preferable preparation form , there are capsules ( soft capsules ) wherein a liquid mixture of the above - described linoleic acid and the pharmaceutically effective ingredients is sealed . tablets are also a preferably preparation form . in forming tablets , the pharmaceutically effective ingredients of the present invention , alone or a mixture of the ingredients , and linoleic acid are adsorbed on a suitable solid carrier and compressed into tablets . if necessary , the tablets may be coated with a suitable coating agent . as the above - described solid carrier , there are illustrated , for example , lactose , starch , carboxymethyl cellulose , dextrin , calcium phosphate , calcium carbonate , synthetic or natural calcium silicate , magnesium oxide , dry aluminum hydroxide , magnesium stearate , sodium bicarbonate , dry yeast , etc . in addition , an antioxidant may be compounded in the preparation . further , those disintegrators , corrigents , sweetenings , colorants , etc . which are used in this field may properly be added unless they accelerate hydrolysis or oxidation of the effective ingredients of the present invention . in a different aspect of the present invention , a process is provided for separating the aforesaid α , β - unsaturated fatty acids from freshwater clams by extraction . freshwater clams to be used in the present invention include corbicula sandai , corbicula leana , corbicula japonica and similar species , which belong to corbiculidae . of those , the use of corbicula sandai reinhardt is preferable . according to the process of the present invention , the whole body or fleshy portions of freshwater clams are used as such or in finely cut pieces , and : ( i ) they are hot - extracted with water at about neutrality , the aqueous extract is concentrated , then the concentrate and / or the separated fleshy portions are extracted with an aprotic solvent ; or ( ii ) they are extracted with an aprotic solvent , and the solvent is removed from the extract and the residue purified to separate α , β - unsaturated fatty acids . with route ( i ), water to be used for hot - extraction with water is preferably in a boiled state . the freshwater clam is preferably extracted without separating fleshy portions from shells . upon extraction , water is used in an amount 2 to 10 times by weight , preferably 4 to 5 times , as much as that of corbicula . this extraction treatment is conducted for 3 hours to 10 hours , preferably 4 to 5 hours . after hot - extraction treatment with water , the aqueous extract is concentrated , and the resulting concentrate and the previously extracted fleshy portions are then extracted with an aprotic solvent . as the aprotic solvent to be used in this extraction procedure , there are used hydrocarbons such as straight - or branched - chain , saturated hydrocarbons , alicyclic hydrocarbons , aromatic hydrocarbons , etc . ; halogenated hydrocarbons such as dichloromethane , chloroform , carbon tetrachloride , etc . ; dimethylsulfoxide ; dimethylformamide ; acetonitrile ; ketones such as acetone and methyl ethyl ketone ; diethyl ether ; etc . of these , saturated chain hydrocarbons are preferable , with n - hexane being most preferable . in extraction procedure , the concentrate of the above - described aqueous extract is preferably in a powdery state , and the extracted fleshy portions are preferably dried and cut into pieces as small as possible . upon extraction procedure , these substances to be extracted are preferably well dispersed in a solvent to realize high contact efficiency with the solvent . for example , the extraction in an ultrasonic wave bath is preferable . with route ( ii ), the same aprotic solvent as with route ( i ) are used . upon extraction procedure , the substances to be extracted are preferably dried and cut into fine pieces . the substance obtained by removing the solvent from the above - described aprotic solvent extract is a mixture containing increased amounts of the aforesaid α , β - unsaturated fatty acids . the mixture has a diabetescuring effect and a lipid metabolism - improving effect in the as - produced crude form . the above - described crude product is further purified . for such purification , column chromatography is useful . as a column support to be filled in a column to be used in column chromatography , silica gel is preferable and , as the eluting solvent , hexane , benzene , ethyl ether , methanol , or benzene - methanol mixture is suitably used . according to a preferred purifying method , the concentrate obtained by concentrating the above - described aprotic solvent extract is placed on the column and eluted with successive , hexane , benzene , ethyl ether , and methanol . the end products of the present invention are eluted with benzene and ethyl ether in a purified form . for isolation of pure products of the compounds of the present invention , it is desirable to further conduct chromatography using successively , hexane , benzene , ethyl ether , and methanol as eluting solvents . the end compounds of the present invention are finally eluted in the ethyl ether fraction described above . additionally , all of the materials obtained in the step of further purifying the above - described aprotic solvent extract show the effect of curing diabetes and the effect of improving lipid metabolism . the present invention will now be described in more detail by reference to pharmacological experiments and examples . two groups of the respective 6 rats described above were used : one group being control group ( a ); and the other being fatty liver - induced group ( b ) to which 2 ml / 100 - g body weight of a 50 % ethanol aqueous solution had been aministered for consecutive ten days by one administration in the morning through a gastric tube . further , to group ( c ) of 5 rats described above were intraperitoneally administered 0 . 5 ml / 100 - g body weight of a solution prepared by dissolving 200 mg of 2 - octadecenoic acid in 5 ml of olive oil ( corresponding to 10 mg of 2 - octadecenoic acid per 100 - g body weight ) simultaneously with administration of a 50 % ethanol aqueous solution in the same manner as with group ( b ). after completion of the above - described administration , rats were decapitated , and blood was collected to measure the amounts of total cholesterol ( chol ), triglyceride ( tg ), phospholipid ( pl ) of blood serum , and the amounts of total lipid , chol , tg , and pl in liver . results thus obtained are tabulated in tables 1 and 2 . table 1______________________________________results of measurement of blood serum lipidstested chol tg planimal group ( mg / 100 ml ) ( mg / 100 ml ) ( mg / 100 ml ) ______________________________________control group 5 . 04 ± 3 . 21 91 . 2 ± 10 . 14 88 . 6 ± 8 . 40 ( a ) ethanol - adminis - . sup . 74 . 3 ± 5 . 58 . sup . a . sup . 141 . 8 ± 10 . 06 . sup . a 94 . 7 ± 13 . 98tered group ( b ) ethanol and 59 . 8 ± 2 . 18 125 . 8 ± 2 . 85 137 . 0 ± 21 . 542 - octadecenoicacid - administeredgroup ( c ) ______________________________________ note . sup . a significant difference was observed over control group ( a ). table 2__________________________________________________________________________results of measurement of liver lipids total lipid amount chol tg pltested animal group ( mg / 100 ml ) ( mg / 100 ml ) ( mg / 100 ml ) ( mg / 100 ml ) __________________________________________________________________________control group ( a ) 36 . 2 ± 1 . 34 . sup . 1 . 93 ± 0 . 13 . sup . 4 . 08 ± 0 . 63 22 . 67 ± 1 . 19ethanol - administered 50 . 1 ± 3 . 43 . sup . a 3 . 21 ± 0 . 24 . sup . a . sup . 9 . 51 ± 0 . 78 . sup . a 23 . 07 ± 0 . 82group ( b ) ethanol and 2 - 38 . 6 ± 1 . 52 . sup . b 2 . 44 ± 0 . 11 . sup . b 6 . 86 ± 0 . 99 24 . 15 ± 0 . 30octadecenoic acid - administered group ( c ) __________________________________________________________________________ notes . sup . a significant over control group ( a ). . sup . b significant over ethanoladministered group ( b ). from the above results , it follows that , in comparison with control group ( a ), ethanol - administered group ( b ) underwent an increase in total cholesterol amount and triglyceride amount in blood serum and total lipid amount , total cholesterol amount , and triglyceride amount in liver with significant difference , but underwent only a slight increase in phospholipid amount in blood serum and liver without significant difference . comparison of ethanol - administered group ( b ) with ethanol - and 2 - octadecenoic acid - administered group ( c ) is as follows . with group ( c ), total cholesterol amount and triglyceride amount of blood serum increased in group ( b ) with significant difference by adminstration of ethanol were observed to tend to be decreased . on the other hand , total lipid amount and cholesterol amount in liver , which were increased by administration of ethanol with signicicant difference , were decreased with significant difference , with triglyceride amount of blood serum and liver being in a decreasing tendency . as is described above , 2 - octadecenoic acid can be said to possess the effect of decreasing the total cholesterol amount and triglyceride amount of blood serum and total lipid amount , total cholesterol amount , and triglyceride amount of liver which were increased by ethanol - induced fatty liver . further , histological examination of tested rats revealed that fat droplets observed with alcohol - administered group rats by dyeing with sudan iii were remarkably decreased with group ( c ) rats . three groups of the respective 8 rats described above were used . one group received ordinary powdery diet ( control group ( a )). another group received the same powdery diet as given to group ( a ) except for adding thereto 1 . 5 g / kg of orotic acid - administered group ( b )). the other group received the same powdery diet as given to group ( a ) except for adding thereto 1 . 5 g / kg of orotic acid and 1 . 0 g / kg of 2 - octadecenoic acid a day ( orotic acid - and 2 - octadecenoic acid - administered group ( c )). the above - described administration was conducted for consecutive ten days with all groups , followed by autopsy to measure liver weight and total lipid amount , chol , tg , and pl of liver . results thus obtained are tabulated in table 3 . table 3__________________________________________________________________________ total liver lipids of weight liver chol tg pltested animal group ( g / 100 g ) ( mg / 100 g ) ( mg / 100 g ) ( mg / 100 g ) ( mg / 100 g ) __________________________________________________________________________control group ( a ) 3 . 61 ± 0 . 077 . sup . 180 ± 12 . 3 . sup . 9 . 14 ± 0 . 177 5 . 34 ± 0 . 843 . sup . 102 ± 3 . 70 . sup . orotic acid - 4 . 57 ± 0 . 322 . sup . a 444 ± 33 . 4 . sup . a . sup . 17 . 71 ± 0 . 898 . sup . a 74 . 48 ± 11 . 7 . sup . a 143 ± 4 . 63 . sup . aadministeredgroup ( b ) orotic acid - and 4 . 01 ± 0 . 130 . sup . b 305 ± 39 . 5 . sup . b . sup . 13 . 34 ± 0 . 941 . sup . b 39 . 8 ± 10 . 16 . sup . b 120 ± 5 . 10 . sup . b2 - octadecenoicacid - administeredgroup ( c ) __________________________________________________________________________ notes . sup . a significant over control group ( a ) . sup . b significant over orotic acidadministered group ( b ) from the above results , it is seen that , in comparison with control group ( a ), orotic acid - administered group ( b ) underwent a significant increase in liver weight and total lipid amount , chol , tg , and pl of liver and that , in comparison with orotic acid - administered group ( b ), orotic acid - and 2 - octadecenoic acid - administered group ( c ) underwent a signicifant decrease in every analysis item . thus , it is clear that 2 - octadecenoic acid is effective against orotic acid - induced fatty liver . three groups of the respective 6 rats described above were used . one group was control group ( a ). another group was fasted for 18 hours , then intraperitoneally administered with 450 mg / kg of d - galactosamine and , after 22 hours , autopsied ( group ( b )). the other group were administered with 1 . 0 g / kg of 2 - octadecenoic acid ( using a preparation obtained by dissolving 2 - octadecenoic acid in ether , mixing the solution with a 2 - fold quantity of lactose , drying and suspending in a 0 . 5 % methyl cellulose aqueous solution ) two hours before the same administration of d - galactosamine as with group ( b ) and , 22 hours after administration of d - galactosamine , autopsied ( group ( c )). enzyme activity of glutamic pyruvic transaminase ( gpt ) and glutamic oxaloacetic transaminase ( got ) of the blood serum obtained by autopsy , liver weight , and total lipid amount in liver were measured to obtain the results given in table 4 . table 4__________________________________________________________________________ liver total lipids serum serum weight of livertested animal group gpt got ( g / 100 g ) ( g / 100 g ) __________________________________________________________________________control group ( a ) 29 ± 5 . sup . 208 ± 19 . sup . 3 . 42 ± 0 . 07 . sup . 4 . 49 ± 0 . 10 . sup . d - galactosamine - 302 ± 87 . sup . a 643 ± 142 . sup . a 4 . 04 ± 0 . 07 . sup . a 5 . 04 ± 0 . 21 . sup . aadministeredgroup ( b ) d - galactosamine - 27 ± 3 . sup . b 214 ± 24 . sup . b 3 . 66 ± 0 . 07 . sup . b 4 . 95 ± 0 . 24 . sup . band 2 - octadecenoicacid - administeredgroup ( c ) __________________________________________________________________________ notes . sup . a significant over control group ( a ) . sup . b significant over dgalactosamine - administered group ( b ) the above results reveal that , in comparison with control group ( a ), d - galactosamine - administered group ( b ) underwent an increase in every measurement item with significant difference . in comparison with this group ( b ), d - galactosamine - and 2 - octadecenoic acid - administered group ( c ) underwent a decrease in gpt and got in blood serum and liver weight with significant difference , with total lipid amount in liver being in a decreasing tendency . from these , it follows that 2 - octadecenoic acid has an effect against d - galactosamine - induced liver trouble . additionally , when two groups of the respective 10 male and female wistar rats ( 200 g ) were orally administered with 5 g / kg and 10 g / kg , respectively , of 2 - octadecenoic acid ( dissolved in olive oil ) for consecutive 5 days , there were observed no abnormality . two groups of the respective 6 rats described above were used . one group was control group ( a ), and the other group was injected with 50 mg / kg of streptozotocin through tail vein to cause diabetes ( confirmed by urinous sugar examination )( group b ). of group ( b ) rats , three rats were orally loaded , after 20 days , with 1 . 0 g / kg of 2 - octadecenoic acid together with diet . after 10 days , chol , tg , pl , and free fatty acid ( ffa ) of blood serum , blood sugar in hunger ( fbs ), and total lipid amount , chol , tg , and pl of liver were examined with every group to obtain the results shown in tables 5 and 6 . table 5 shows that , in comparison with control group ( a ), streptozotocin - administered group ( b ) underwent a slight increase in tg and ffa , and a significant increase in chol , pl and fbs . 2 - octadecenoic acid - administered group ( c ) underwent a significant increase in chol in comparison with group ( a ), and underwent a significant increase in fbs in comparison with group ( b ). table 6 shows that , in comparison with control group ( a ), streptozotocin - administered group ( b ) underwent an increase in total lipid amount , a decrease in chol , and a significant increase in tg and pl . on the other hand , chol , 2 - octadecenoic acid - administered group ( c ) was lower than that of group ( a ) or ( b ), and tg was lower than that of group ( b ). total lipid amount and pl of group ( c ) were significantly lower than those of group ( b ). from these , it follows that 2 - octadecenoic acid has the effect of improving or curing liver function or the effect of decreasing blood sugar against diabetes . table 5__________________________________________________________________________tested animal chol tg pl ffa fbsgroup ( mg / dl ) ( mg / dl ) ( mg / dl ) ( meg / l ) ( mg / dl ) __________________________________________________________________________control 61 . 2 ± 2 . 06 . sup . 172 . 3 ± 17 . 12 104 . 0 ± 3 . 33 0 . 75 ± 0 . 066 121 . 5 ± 5 . 06 . sup . group ( a ) streptozotocin - 78 . 9 ± 3 . 94 . sup . a 190 . 4 ± 20 . 29 . sup . 136 . 4 ± 13 . 54 . sup . a 0 . 83 ± 0 . 059 464 . 4 ± 41 . 41 . sup . agroup ( b ) streptozotocin 74 . 3 ± 6 . 35 . sup . a 147 . 4 ± 15 . 20 94 . 1 ± 15 . 48 0 . 74 ± 0 . 098 190 . 9 ± 67 . 51 . sup . band 2 - octadecenoicacid - administeredgroup ( c ) __________________________________________________________________________ notes : . sup . a significant over control group ( a ) . sup . b significant over streptozotocinadministered group ( b ) table 6__________________________________________________________________________tested animal total lipid chol tg plgroup in liver ( mg / g ) ( mg / g ) ( mg / g ) ( mg / g ) __________________________________________________________________________control 42 . 43 ± 2 . 849 2 . 64 ± 0 . 301 5 . 08 ± 1 . 033 21 . 86 ± 0 . 480 . sup . group ( a ) streptozotocin - 48 . 12 ± 2 . 836 2 . 57 ± 0 . 145 . sup . 7 . 61 ± 0 . 901 . sup . a 28 . 04 ± 0 . 688 . sup . aadminsteredgroup ( b ) streptozotocin . sup . 37 . 32 ± 2 . 768 . sup . b 2 . 23 ± 0 . 097 5 . 53 ± 1 . 036 23 . 76 ± 1 . 452 . sup . band 2 - octadecenoicacid - administeredgroup ( c ) __________________________________________________________________________ notes : . sup . a significant over control group ( a ) . sup . b significant over streptozotocinadministered group ( b ) 800 kg of corbicula were boiled in water , and an aqueous solution was spray - dried to obtain 6 . 4 kg of aqueous extract powder , with fleshy portions being separated from shells and dried by hot air to obtain 11 . 7 kg of a dried product . these were extracted with n - hexane in a supersonic wave bath to obtain 1 . 4 kg of a viscous , blackish - brown oil . this oil was subjected to column chromatography ( using 700 g of 60 to 80 - mesh silica gel filled in a glass tube of 5 cm in diameter and 100 cm in length ). as eluting solvents , 1 liter each of hexane , benzene , ethyl ether , and methanol were used in this order . a latter 200 ml portion of benzene fraction and a former 200 ml portion of ethyl ether fraction were collected . they were concentrated to obtain 700 g of an oil , which was subjected to the same column chromatography as described above , followed by concentrating a benzene fraction and an ethyl ether fraction in the same manner as described above to obtain 400 g of an oil . this oil was further subjected to column chromatography using hexane , benzene , ethyl ether , and methanol in this order as eluting solvents . thus , 21 . 5 g of an oil containing compounds forming color by irradiation with uv light ( observed on tlc ) was obtained from the former portion of the ethyl ether fraction . this oil was methylated with diazomethane in a conventional manner , and the resulting methyl esters were analyzed by gas chromatography to obtain a gas chromatogram constituted by four main peaks . compounds corresponding to respective peaks were gas - chromatographically collected and analyzed through infrared absorption , nmr , amd mass spectrum and , as a result , the thus obtained data were found to completely coincide with the spectral data of separately synthesized methyl 2 - tetradecenoate , methyl 2 - hexadecenoate , methyl 2 - octadecenoate , and methyl 2 - eicosenoate . additionally , these acids were solids at room temperature ( 26 ° c . ), and are comparatively stable compounds though being modified by solvolysis or oxidation .
a medicinal composition which comprises a compound of the formula : ch . sub . 3 nch ═ chco . sub . 2 h wherein n represents 10 , 12 , 14 or 16 , or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier ; a method for testing diabetes or improving lipid metabolism by the administration of said composition to a human being ; and a process for the separation of said compounds from freshwater clam which comprises subjecting freshwater clam to the extraction with hot water and / or an aprotic solvent and then purifying the resultant .