滋賀大学学術情報リポジトリ

セタシジミの生化学的研究

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Biochemical Studies on “ Setashijimi” （Corbicula sandai Reinhardt）

T． Hori   Of the biochemical studies on mussels， a study of the seasonal variation of chemical components of “ Magaki ” （Oslrea gi．aas Thunberg） by Masurnoto and his co−workeri）， and that of the mechanism of shell−for皿ation of“Akoyagai” （a mother mussel of pearl）， with Ca‘5 labeled calclum carbonate by Tanaka and his co−woker2）， have hitherto been prominent， while detailed studies of individual co皿ponents of the mussel meat have been scarcely done． Although the mussel lipids， one of the lndividual components， are said to contain complex lipids more than fish lipids do3）， and thought to play an i皿portant role in biochemistry， there has been no research but those of the metabolis皿and of the component fatty acids by a few persons．475）   In order to execute the studies on fresh−water皿ussels， especially on their lipids， “ Setashiji’mi” （Corbi．fiula sandai Reinhardt） was here used as experimental materials， because there have been few reports of the fresh−water］皿ussels， and our university being located by Lake Biwa， the fresh mussels can be easily gathered．   The author at first investigated the seasonal variations of chemical compo− nents of the mussel rneat， because he doubted whether the data obtained from 皿arine・皿ussels by other persons can be applied to the case of“Setashijimi” which Iives in different surroundings froln the forエner． Next， for the purpose of elucidating especially characteristics of the lipids， one of the chemical com− ponents， the further investigations of the lipid co皿ponents were carried out・ These results will be described in this paper．

GENERAL

  1． The Seasonal Quantitatiye Variations of Chemical Components of the Mussel Meat．   Owing to the diMculty of cultivating “Seta− shijimi ”，6） the natural mussels gathered from the location as shown in Fig． 1 were used for this study． The gathering location， which is near the railway bridge over the Seta River， has the follow− ing environment 7）： the velocity of stream 35 cm．／ sec．， pH of water 7．0−v7．4， the depth of water 2−v 4m， and contents of organic substances s mg．fl． （mean values through a whole year）． The plank− ton there breedsエ11uch from May to September while little from January to February． As there live much “ Setashijimi ”， several hundreds of the mussels were gathered at random at the begin一 Fig． 1．   Sketch of the gather− ing location of “Seta− shijimi．” X： gathering location．

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ning of every month between 4 p。m． and 5 p．皿．， and from them 100 to 110 pieces of the adult mussels having 1．5 to 2．Ocm． of shell−lengthS） （Fig． 3） were selected， and washed thoroughly with water． After they were dried in ordinary room and weighed， they were separated into the shell and the meat containing ligaments by quick boiling with 50ce． of water for two minutes．   In shelling the meat from the mussels， the author adopted the boiling method in place of the common dissecting one， because the mussel was too small to excise the meat in the original for皿， and apt to be miserably di＄figured． Thereby， the components eluded in the juice by boiling were determined， and the following results were obtained ： the amount eluded was attained to 2．5 per cent of the tota1 meat and its components consisted mainly of 35．2 per cent of proteins， 48．9 per cent of glycogen． O．62 per cent of lipids and 13．7 per cent of ashes． They corresponded to O．9 per cent， 1．2 per cent， O．1 per cent and O．3 per cent of the whole rneat respectively， and these values seemed too small to give much error to the whole corr．］．ponents cf thc ir．］tussel rr．iieat． The juice compositions， therefore， eluded from the mussels can be neglected．  The shell and the meat thus obtained were separately transferred on filter paper， and after being carefully dried at a room temperature for about an hour and weighed， the adequate amount of the meat was divided into four parts to deter− mine the contents of total nitrogen， glycogen， ］ipids， moisture and ashes． The roo皿temperature and the hydroscopic degree in summer differ markedly from those in winter， so that the author adjusted the drying time to keep the similar conditions．   The weight of the shell， the meat， the dried lneat， the weight ratio of the dried］皿eat to the shell and the contents of chemical components of the mussel meat were determined monthly．   The data are shown in the following Table 1， and the variation curves of the chemical cornpo− nents and that of the above ratio （the author named this ratio the fatness degree） are exhibited in Fig． 2 with the curve of an incubation degree representing the gonad ripness of “Setashijimi ” examined by Furukawa．9）   In the seasonal variations of the rneat as shown in Fig． 2， the pro；ein and the glycogen exhibit the variations remarkably， while the lipid does slightly． That is， the protein increases from       Table 1． Contents of chemical components of the mussel rneat．

Mean

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Percentage

Month

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Protein

GlycogeR

工ipid

Ash

Total

Nov． 2．74 0．31 0．12 4．38

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17ユ 6．5 2．8 2．6 100．6 Dec． 2．93 0．30 0．11 3．67 68．7 17．6

Z5

3．2 3．0 100．0 Jan． 2．94 0．29 0．11 3．76 69．9 17．4 7．9 3．3 1．9 100．4 Feb． 2．71 0．34 0．11 4．05 71．6 17．0 8．3 3．3 1．1 101．3 Mar． 2．62 0．26 0．09 3．43 70．5 16．7 8．8 3．4 1．5 100．9 Apr． 2．84 0．48 0．14 4．93 69．5 16．5 9．8 3．8 1．5 101．1

May

2．77 0．80 0．24 8．67 69．4 16．2 10．0 4．2

L9

10L7

Jun． 2．59 0．49 0．20 ス74 70．6 15．9 7．5 4．5 3．1 101．6

JuL

2．75 0．50 0．16 5．81 71．1 14．5 5．5 4．1 3．5 98．7 Aug． 2．81 0．46 0．14 4．98 72．7 13．8 4．8 4．2 2．6 98．1 Sep． 2．72 0．28 0．08 2．94 73．4 14．1 5．5 3．2 2．2 98．6 Oct． 2．69 0．39 0．11 3．62 71．9 15．3 6．2 2．7 1．9 98．0 August（13．7 ％ ； the minimum content）to December （17．6％； the maximum content）， and then gra一 dually decreases during the following January to May， and suddenly falls from June to August．

セタシジミの生化学的研究

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Glycogen

Lipid Incubation degree Fatness degree  Are of oblique lines shows the generative time of “ Setashiji’mi．” The glycogen shows its皿inimum content（4．8％） in August as the protein； from the following month on it continuously increases till next May， but after reaching to the maximum content （10．0 ％） in May， suddenly decreases just in the case of the protein．   Contrary to the remarkable variation of the above coエnponents， that of the lipid is 皿ild；it reaches its maximum content （4．5％） in June a little later than glycogen does， and decreases to reach its minimum （2．7％） in October．   These results of‘‘Setashiji皿i”10）beingco皿pared with the seasonal variations of “ Magaki ” studied by Masumotoi） and of “Akoyagai” by Tanaka！）， it is found that the protein of the for皿er exhi− bits more variation than the latter two， and that the rise and fall of the glycogen of the former rese皿bles that of the others， but contrary to our expectation the content of the former at the minimu皿period does not fall into a trace while the others do；the Iipid of“Setashiji皿i”shows less seasonal variation than those of “Magaki” and “ Akoyagai”， and the former lipid reaches its maximu皿content olleエnonth later than the glycogen does， which agrees with the case of “ Magaki ”， but is reverse to that of “ Akoyagai．”   The weight ratio of dried meat to shell， the fatness degree named by the author， ls considered to indicate the activity of the mussels’ life． The fatness degree， as shown with dotted line in Fig． 2，reaches to a■ninimu皿（3．4％）during January to February， then suddenly rises to a large皿axi− mum （8．7 ％） in June， and quickly falls down to another minimum（3．0％）in Septe皿ber． Then it gradually rises to a small皿axi皿um（4．4％） in November， and falls again． From the above variation of the fatness degree， it is supposed that “ Setashijimi ” has the two emaciating periods， the one is January to February and the other about September．   When the variation of this fatness degree are compared vv’ith the incubation curve which rises from t’he middle of May to that of August， some interesting facts which attract our attentions are found． “Setashij iTni” grows from September to February， during which it gradually fattens its emaciated body caused by ovulation and ejacula− tionee during the period of discharge of sexual products （summer） ； it fuliy developes by being well nourished during the period of growth （au− tumn to winter）； it prepares itself for the gonad ripening （spring） and consumes itself during the iollowing time （summer）． The small maximum of the fatness degree in November will be due to that “ Setashijimi ” ingests much food materials ee The ovulation and the ejaculation take place in waterS’6）

before winter hibernation．   The ash content （Fig． 3） represents seasonably the considerable variation having two maxima in December （3．0 ％） and July （3．5 ％）， two minima in February （1．0 ％） and October （1．9 ％） respect− ively， but the ash content of “Setashijimi” （1．0 ％・一一3．5％） is less than a half of those of “Ako− yagai” （7．5％一一12．1 ％） and “Magaki” （5 ％一一8％）． This remarkable difference of the ash contents is supposed to co皿e from the different circum− stances that the former lives in the fresh water and the latter in the sea． ↓口①省oQ ，

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   一         一       一       Fig． 3． Variation curve of the ash．   Areas of oblique lines show periods of forming the resting zone of “Setashijimi．”   Such bivalves as “Setashijimi” make a con− c）ntric circular resting zone on the surface of shells in the course of their growths， and the resting zone in the case of“Setashijimi”appears twice in a year independently of the size of the shells． The one is formed in June （the obstacle ring）， the other from December to February （the wiriter one）， and the latter is said to be clearer than the former． The color of these zones is greenish brown， and the distance of the ad− jacent zones， the H−h as shown in Fig． 4， which indicates the degree of shell growing， is about 4．一6mm． at the longest．ii）

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Fig． 4． Sketch of the shell．      H： Shell−height      L： Shell−length       circular lines exhibit the resting   It is naturally supposed that there is a bioehemi− cal correlation between the variation of ash con− tent of the皿ussel meat and the degree of shell Oく● Photo． 1． 灘i、

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陣 ，．， growing， and in practice from analytical results the both periods of rnaximum content of the ash are found to coincide well with those of forming− P111        噛   ． the resting zone， which is an interesting subject which attracts our attention （Fig． 3）．   In short， the author has studied on the quanti− tative variations of chemical components of “ Seta− shijimi” and has discussed on the results． ln order to make further discussion possible， how− ever， he ha’刀@to proceed to the study of the quantitative variation of organic and inorganic constituents of the above components that play important role in the biochemical change occur− ring in “ Setashijimi：’   He， therefore， began at first the investigation of qualitative variations of the lipid and the in− organic constituents of the meat， while those of the protein and the glycogen will be done in the near future．   2． The Seasonal Qualitatiye Variations of Lipids and lnorganic Constituents of the Mus− sel Meat．   In order to observe the qualitative variations of the lipids， “ Setashijimi ” was monthly gathered from March， 1953， to February， 1954， and treated by the procedures described later （p． 120） to pre− pare the lipids．i2） Of them the acetone−soluble lipids were used for this observation， while the acetone−insoluble ones prepared at the same time， owing to the scarcity of them， were left untouched which will be studied in the future． The char− aeteristics of the acetone−soluble and 一insoluble lipids prepared in June， are cited as an instance in the following table． t

セタシジミの生化学的研究

Fraction

APP． Iod． v． Sap． v． N（％） P（％） CorLtent （％）

Acetone−soluble Iipids

Dark：brown

157．4 151．5 0．09 0．19 73 Acetone−insoluble王ipids

Dark：brown

120．0 183．9 1．24 3．60 27   As seen in the above table， the nitrogen and the phosphorus which seem to come from phos− pholipids （described later） are present in the acetone−soluble lipids also． The lipids， therefore， are considered to contain about 5 per cent’k of phospholipids in adctition to simple lipids and un− saponifiable matter．  Their appearances， refractive indices， acid values Table 2． Characteristics ef the acetone−soluble lipids and the lipid acids．

Month

Mar． Apr．

May

Jun． Ju1． Aug．

Lipids APP。 @   nD `cid value rap． value hod量ne valu［e   GB @1．4672 P2．4 P56．4 P55．2   GB @1．4725 P1．0 P47．4 P66．6   GB @1．4746 @8．3 P46．0 P68．1   DG @1．4749 @6．5 P51．5 P57．4   DG @1．469エ @5，7 P56．4 P53．8   DG @1．4759 @9．5 P49．6 P43．0 Acids APP． @   nD meutral． value hodine value   Gb @1．4762 P95．0 P76．0   GB @1．4622 Q09．0 P81．0   GB @1．4592 Q04．4 P70．7   GB @1．4595 Q08．8 P67．7   DG @1．4613 P96．2 P60．1   DG @1．4609 P90．9 P54．5 H−Content（％ ar−Content（％） 13．6

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L圭pids APP． @   11D `cid vahle rap． value hodine value   DG @1．4704 @4．5 ?T2．0 P43．6   Gb

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  H−Content ： Content of highly unsaturated fatty acid． Br−Content ： Content of bromine． U−Content： Content of unsaponifiable matter． GB： Greenish brown． DG：Dark green． RB：Reddish brown． Yo：Yellowish orange． Ro： Reddish orange． ac It is a calculated value assu皿ing that the phospholipids contain 1．8 per cent of nitrogen and 4．O per cent of phosphorus．

saponification values and iodine values were determined． Moreover， after saponifying the lipids with alcoholic potash， the characteristic of the lipid acids recovered from the soap solu− tion by acidification were deter皿ined。 These determinations were executed by the usual pro− cedures used for identification of fats and oils． The contents of highly unsaturated acids were also determined by the method of bromination． Besides， as to the unsaponifiable matter， its con− tents， appearances and iodine values were observ一 ed． All these results are shown in Table 2．   As seen from the above table， the variations of the refractive indices and of the acid values show no seasonal regularity， and the appearances alter from a deep green （July to October） to a greenish brown （November to June）， but the reason can not be understood． Since the other properties except the above mentioned ones seems to show some seasonal variations， especially be− fore and after the period of the generative time， to make them more clearly the following figures

Fig． 5． Fig． 6．

Variations of properties of the lipids and the lipid acids．

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セタシジミの生化学的研究

are shown （Fig． 5N6）．  』The content of unsaponifiable皿atter as shown in Fig．6increases fro皿March to May；then it decreases till the end of the generative time and after the time it gradually increases again． The sudden decrease of the unsaponifiable matter during the generative time is probably due to the discharge of sexual products． The unsatura− tion degree of the iipids which is expressed by iodine values （Fig． 5）， lncreases from March to May and decreases from August to September， and after October increases gradually again． lt see皿s that the great difference of unsaturation degree between pre一 and post−generative times is due to the transformation of the unsaturated constituents in the lipids into the other saturated one or the consumption of themselves． An un− sa加ration of lipids being supposed to come chiefly from the ethylenlinkage of the lipid acids and the unsaponifiable matter， the author tried to decide on which the ethylenlinkage is based， on lipid acids or on unsaponifiable matter by coxn− paring the rise and fall of iodine value between the both．   It was found that the unsaturation of the “Setashijimi” lipids chiefly depends on that of the lipid acids， because iodine value of the acids showed a remarkable variation before and after the generative time， while that of the unsaponi− fiable matter did not show so remarkable in the whole year as shown in Fig． 5． Moreover， from the fact that the variation curve of contents of highly unsaturated acids roughly resembles that of unsaturation of the acids （Fig． 5）， the rise and fall of unsaturation of the lipids is presumed to depend on that of contents of highiy unsaturated acids after all． The unsaturated acids， therefore， are supposed to contribute to the generative pro− cess of “ Setashijimi ”ee   The color phase of unsaponifiable matter （Table 2） alters from a reddish orange to a yellowish orange at the end of the generative time． From this （bD W絹．剃医︶のぢ①コ戯お8Q。帽器bD﹄8Hも娼g80 fact， it is anticipated that the reddish animal pigment is a substance correlative to the genera− tive process of “Setashijimi，” though it is chemi− cally unknown what this pigment is．i3）  For the purpose of observing the qualitative variations of inorganic constituents of the mussel         ’ 皿eat， the meat obtained from 100 pieces of the selected mussels was burned in an electric furnace， and the ashes obtained were analysed by the usual procedures． These results are shown in the following Fig． 7， where the contents of sulfate， 一・＝acO一・一Gpt・’

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   一 一      一       ． 一   Fig． 7． Variations of inorganic constituents of the mussel meat． ac Prof． Toyama （Nagoya Univ．）， in his letter to the author， stated the unsaturation degree of animal lipids is generally supposed to increase before their gonad or when they ingest much food materials，  （June 1953）．

iron， copper， manganese and alminium in the ashes were too small to determine their amounts， so that the analysis of them were omitted．   As shown in the above figure， the inorganic constituents of the mussel meat are composed mainly of phosphate， silicate and calcium oxide， and the contents of these show that they under− go the remarkable variations seasonably． That the silica reaches to its maximum during the surnmer time is probably due to that “Setashi− jimi ” ingests much diatoms in the season，55） and the calcium oxide rises to its maxi皿um at the periods of forming the resting zone． Judging from the faet that the shell contains much calc− ium as its carbonates （Table 3）， the calcium of theエnussel meat is considered to contribute to the for皿ation of the shell． Although at first sight it seems strange that“Setashiji皿i”contains much of phosphate in its meat while none in its shell， it is realized from this fact that the phosphate is supposed to play a good role in phosphometa− bolism， while no used for the construction of the shell．   In the qualitative observation of the lipids and Table 3． Composition of the shell

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e the inorganic constituents of “Setashijimi，” the author noticed that the former shows a conside− rable variation in their qualities， particularly， in an unsaturation degree and a color of the un− saponifiable matter， and that the latter which chiefly contains silicate， phosphate and calcium oxide also shows remarkable variations． He， therefore， has mentioned above his view concern− ing the biochemical correlations suggested in these qualitative variations．   In the above study， ‘’Setashijimi” has been found to contain a variety of lipids besides simple lipid＄． These seem to lnclude complex lipids such as phospholipids， derived lipids such as sterols． Accordingly， in order to elucidate all the biochemical functions of the lipids， more advanced knowredge about them has been necessitated in the biochemical studies on “Setashijimi．”   3． The Lipids．   It is generally said that mussel lipids， when compared with fish lipids， contain more unsapo− nifiable matter and the acetone−insoluble lipids，3） and the unsaponifiable 皿atter of the former， especially the sterol has widely been investigatedi4）．   The several of new sterols such as corbisterol and meretristerol have been discovered， which has attracted our interests． Contrary to the above case） the acetone−insoluble lipids， the complex lipids， have scarcely been researched and the only outline of studies on t’he lipid acids except studies of“Magaki”by Masumoto，1のand of“Mashili皿i” and “Hokkigai” by Toyarna has been reported．   A） Fatty components of the lipid acids   As previously mentioned， in order to elucidate the biochemical functions of the皿ussel lipids which has been seem in their qu21ities to． h2ve 2 close relation to the generative process， it is necessary to investigate the component of the lipids in details． The author at first studied on the lipid acids， because to know the characteris− tics of the fatty acids common to the lipid com− ponents was better way to the further inve3ti− gation．18）   The earlier investigations on component fatty acids of mussels have been done within the scope of marine mussels except a few cases， and have been very few． Tsuli皿oto and I（oyanagi19）stated the presence of palmitic acid as a “solid” acid constituent， and of some highly unsaturated acids and oleic series acids of Ci6 and Cis as a “ liquid ” acid constituent， in squilla oils． According to the same authors’reports，20）there are also皿uch of the high！y unsaturated acids， in oils of “Asari，” “Shijimi，” “Karasugai，” “Hamaguri，” “｝lotate− gai，” “Murasakiigai”L’i） and “Sazae”． Ueno and Kuse2L’） indicated that in “Hokkigai” oils highly unsaturated acids are much contained and皿oroc一       ’ tic acid is a chief component． Masumoto showed that the component acids in “Magaki” fats are

セタシジミの生化学的研究

Ci4 acids （14 ％）， C，， acids （23 ％）， C，， acids （23 ％）， C2， acids （33 ％）， C，．． acids （16 ％） and C2， acids （1 ％）， and that above all the C22 acids contain 70 per cent of tetraenoic aeids． “Ma− shijimi”（Corbicula leana）fats were exa皿ined by Toyama and his co−workers，i7） who found that there are palmitic acid，皿yristic acidec’ and stearic acid， as a saturated acid constituent （！6 ％）， and zoo皿aric acid and C20（F，）acids， as an unsaturated acid constituent．   “Setashijimi” used for this experiment was shelled by the boiling method as already described， after dried in 95C・ylOO＝C， pulverized， and then treated with ether to extract the lipids． The lipids 6btained showed the following constants： n“c） 1．4774， acid value 13．3， saponif． value 152．4 and iodine value 142．9．   After saponifying the lipids with alcoho1ic potash the lipid acids recovered from the resulting soap solution showed a greenish brown solid （at 180C）， neutral． value 192．2 and iedine value 142．9． The acids were converted into their metal salts． and       ’ then separated the “solid” acids （27．4 ％）， the “ lipid ” acids （40．1 ％） and the highly unsaturated acids （32．5 ％） by treating the salts with alcohol and acetone， and each of the three free acids， be− ing reconverted into the皿ethyl esters was frac− tionally distilled under 10mm， pressure of皿ercury． Of these distillations． the results of t’he “solid ”       ， acid esters are shown in the following Table 4． Table 4． Fractional distillation of methyl esters of the “soild ” acids． Esters

Acids

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一 4 158∼165［L4300        226．3 8．2 240．9 _54∼55 84．0∼84．5 Myristic 5 _165∼180 1．4315 212．1 3．1 231．3 _47∼48

Myristic 6

oalmitic 4

6 180’一190 1．4327 203．5 9．5 220．9 60．5∼61．5 88∼89 Palmiticrtearic 7  卜

Residue

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一 一   In the above table， the left part shows the con− stants of the ester fractions obtained by the dis− tillation， and the right does those of acids recovered from the fractionated ester． From 2nd and 4th fractions lauric acids and］皿yristic acids were isolated and confirmed respectively， and each of 3rd， 5th and 6th was suppcsed from their physical constants to be a mixture containing two adj acent acid esters． For the purpese of examining these mixtures， they were converted into the free aeids， the constants of which were determined． Their content ratios calculated fro皿the neutra1． values and melting pointsL’3） were shown in the right hand column ef Table 4， and the mixed acids were found to be tauric and myristic， myristic and palmitic， and palmitic and stearic acids Te− spectively．  Table 5． Fractional distillation of皿ethyl esters of the “liquid” acids． Fr．

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Residue

1．4571 1．4582 1．4592 1．4662 1．4696 1．4711 Sap． v． 230．6 207．6 190．1 Iod． v． 108．3 107．0 100．8 120．1 137．5 127．2 Yield （g） O．8 3．2 2．5 5．6 2．5 5．3 2．0 2．1 ee A little later than the author’s report， Y． Toyama showed the presence ot this acid in the lipids  of Corbicuta leana．i6）

  The results of the “ liquid ” acid esters fraction− ation are as follows （Table 5）．   It was presumed from the constants of acid esters in the above table that 2nd and 4th frac− tions consist chiefly of the esters of oleic acids series of Ci4・一一Cie and that in 5th， 6th and 7th ones there are the esters of Cis （Fi・一一vF2） acids． The latter fractions， therefore， were combined together， saponified， and brominated in petroleum ether． The insoluble bromides produced contained 50．01 per cent of bromine， and the recovered productsE4） from the bromides soluble in petroleum ether showed the constants of nBe 1．4630， neutral． value 195．1 and iodine value 112．5． From these constants， the above fractions were supposed to contain chiefiy of Cl， （F，・vF2） acid esters， lower unsaturated esters．   Moreover， the results of the highly unsaturated acid ester fractionation are shown in Table 6．   Of the fractions in Table 6． 2nd fraction was       ， Table 6． Fractional distillation of methyl esters of the highly nusaturated acids． Fr．

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Residue

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（Br． ％） 67．35 69．66    M． P． of bromide（Dark） 210ec 213℃ distilled repeatedly and a fraetion boiling at 2050 N2150C under 15mm． pressure of mercury was collected． Esters of this fraction showed the following constants： nBe 1．4840， saponif． value 197．0 and iodine value 341．5， and their bromides contained 68．31 per cent of bromine， and changed at about 215℃ into a tarry matter． From the fact that these are very close to the constants of CiB （F4） acid ester，L”5） the above esters were considered to be moroctic acid ester． ln addition， it was supposed from the constants of the higher boilling fraction than 4th one in Table 6 that highly unsaturated acid esters of C20 and Cn are present in their fractions．［6）   In short， the methyl ester into which were converted the lipid acids derived from the “ Seta− shijimi ” lipids were fractionally distilled， and from the results of identifications of these esters it was found that there are lauric， myristic， palmitic and stearic acids， as a “saturated” acid constit− uent， and that in the “unsaturated” acids there are C］4 （Fi）， CiG （Fi）， Cis （Fi）， linoleic and linolenic series acids， as a lower unsaturated constituent， and CiB （moroctic acid）， C20 （F4） and C20 （F4−s） acids as a highly unsaturated acid constituent． Of these acids， lauric and myristic acids has been only presumed to be present but not confirmed in mussels， and highly unsaturated CエR∼C20 acids are much contained while the C22一．C2． acids 1ittle in “Setashijimi ”， which are reverse to those of marine fish and “Magaki”． The other acids， however， did not seem to be so different frorn fish lipid acids．   B） Preparation of acetone−solub）e a・nd−insol− uble lipids．   Next the author intended to investigate the complex lipids of “Setashijimi” in details． ln the previous studies on seasonal variation of the mussel meat， much phospholipids with simple lipids and sterols were already found in the lipids． Therefore， in order to know the general charac− teristics of these lipids cornponents， the lipids obtained from the mussels were separated by the procedures as summarized in Table 7， and t’hen investigated respectively．   The outline of the separating procedures is as follows． The mussel meat prepared from “ Seta− shiji皿i”gathered in June，1953， was minced and immediately soaked in acetone to extract the lipids． The acetone solution obtained was de− canted， and fresh acetone was added to the residue． The operations were repeated untill the acetone

セタシジミの生化学的研究

Table 7． Sehema of separating procedure of the lipid components and their characteristics．       Mussel meat （］3．3 kg．）        皿inced and extratced        with acetone．   L

Extract

   added ether．    1 Aqueous solution     1

Residue

     powdered and ex−      tracted with ether．          1 Supernatant （A） Yield： 357．3 g． App．： Dark brown       viscous liquid lod． v．： 159．O Sap． v．： 163．2 Reichert−Meiszl v．： 1．8 Polenske v．： 1．3          ，      l Extract （B）   Yield ： 135．4 g．  App．： Brown viscous liquid   Iod． v．： 130．7  Sap． v．： 177．6      t extracted with acetone and cooled in an ice box overnight．   l

Residue

         l Soluble materials （PART 1）   Yield： 406． 9 g （70 ％）   App．： Dark brown      viscous liquid   Iod． v．： 137．2   Sap． v．： 149．2    N： O．23 ％    P： O．37 ％ Liebermann reaction：十         1   1nsolube皿aterials Yield： 85．5 g． （30 ％）       1 dissolved in ether （1 1．）        and in petroleurn ether        （500 cc．）， and cooled in        an ice box． overnight．       i Supernatant （PART II）   Yield： 82．0 g． App． ： Brown viscous liquid   Iod． v．： 120．O   Sap． v．： 182．9     N： L24 ％     P； 3．20 ％        treated with        95 ％ alcohol．       L Soluble materials （PART Ila） solutions no more colored． After distilling off the acetone from the solutions， the extract was treated with ether to get rid of the accompany− ing substances dissolved in water． The water layer separated from the ethereal solution was removcd off， and the remaining ethereal solution， after dried over sodium sulfate， was distilled． The residue thus obtained， A， was a greenish brown viscous liquid． On the other hand， the 皿eat which was insoluble in acetone was smashed and then treated with ether repeatedly． The ethereal solution accumulated， after it had been dried over sodiu皿sulfate， was distilled and obtain− ed the residue， B， which was a brown viscous liquid． The A and B were combined together， and then subjected to the continuous extraction with dry acetone by Sudo’s liquid extractor． The acetone solution obtained was placed in an ice box for one night． There appeared the precipitates， which were separated from acetone solution by filtration． T． he acetone ＄olution was distUled off，       l Insoluble materials       1 Precipitates （PART III）   Yield： 3．8 g． ApP．：White a皿orphous皿atter   Iod． v： 52．O   M． p．： 2000c （dec．）    N： 3．54 ％    P： 4．2e ％ Liebermann reaction： 一     Molisch reaction： 十    Anthrone reagent： 十   （PART Ilb） and the acetone−soluble matter， Part 1， was thus obtained． The above acetone−insoluble rnatter separated was dissolved in et−her， and then to the mixture was added petroleum ether， kept in an ice box overnight and separated into the white amorphous precipitates， Part III， and the solu− tional part． From the latter， after driving eff the solvent， the residue， Part II， was treated with 95 per cent alcohol， and the alcohol−so− luble part， Part Ila， and the alcohol−insoluble one， Part工lb， were obtained． The general char． acteristics of these parts are shown in Table 7．  From the properties of each of these parts， it was considered that Part 1 consists chiefiy of simple lipids and an unsaponifiable matter， Part II phosphoiipids， and Part III cerebrosides respect− ively．  C） The acetone−soluble lipids： the compQ− nent fatty acids of the simple lipids and the existence of corbisterol．  ？． art I was used to determine the component

Table 8． Fractional distillation of the component acid esters．        B． P． Fraction ！c／s−ih’ ih Hgl nB’2 Sap． v．1 lod． v． Obs． v． Calc． v． （g） 1 （％） （g） 1 （％）    1 @  2 @  3 @  4 @  5 @  6 @  7 @  8 @  9

qesidue

  ∼137 P37∼144 P44∼158 P58∼165 P65∼179 P79∼186 P86∼204 Q04∼211 Q11∼229 Q29∼ 1．4370 P．4360

k4370

P．4390 P．4476 P．4518

k4603

P．4754 P．4839 246．0 Q32．8 Q21．0 Q07．0 P98．7 P90．2 P80．5 P71．2 P68．3   4．1 P3．9 Q1．7 R3．2 X0．7 P29．8 P90．5 Q87．0 R02．0 黶@一 2．8 R．4 P8．8 S4．7 S6．1 S3．4 Q9．4 Q5．1 P1．3 P5．0 1．2 P．4 V．8 P8．6 P9．2 P8．1 P2．2 P0．3 S．7 U．5  2 P4 V7 W0 S6 @6   1 @ 6 R4 黶@一 R5 Q0

@3

fatty acids， of which the simple lipids are com− posed， and a AS，’ sterol， the presence of which the absorption spectrum of the part exhibited．27）        ln 君の口O唱罵O冒OO o，s a 0ユ 親0 260 2お 30D Sto Wave length（rny）   Fig． 8． Absorption spectrum of Part 1 in alcohol solution．   For the purpose of separating the Part 1 into the component acids and the岨saponi且able matter， it was saponified with alcoholic potash． From the saponifiable part the component acids were recovered， and from the unsaponifiable part sterol having reddish orange color was obtained． The former acids were converted into the methyl esters having iodine value 150．9， saponif． value 207．6 and nfiO 1．4609， and they were fractionally distilled by Whitmore−Lux’s apparatus2S） （Photo． 2） and the following nine fractions were obtained （Table 8）．   As shown in the above table， the boiling points of 2nd， 4th， 6th and 8th fractions obtained as the main fractions， were 137“．一v144eC， 1580N1650C， 17ge 一一186℃ and 204e・一一2110C and these boiling points corresponded to those of Ci4， Ci e， Cig and C20 acid methyl esters under 5m皿． of mercury， and the odd number fractions （lst， 3rd， 5th， 7th and 9th fractions） were supposed from their physical Photo． 2． The distilling apparatus．   Single−turn helices of cuperous wire m皿．）were used to pack the column． （diam． O．9 t

セタシジミの生化学的研究

o

L

 o @    モ ｮkhγ。釈e断e

 A

  ノ @ B c Agbegtos C 02’一一）V Z ，e一一一   Wate r ．一・一一jF

6ecm

rf

E

wrNsl i dax

F

トジ「b1へ∼piYαtor S（iddx

G

 Sketch of the distilling apparatus． A： lnside tube 15mm． B： Middle tube 25 mm．， winded with   15 ft． of nichrome wire （No． 22），  wider at top than bottom． C： Outside tube 33 mm．， winding with   asbestos cord． D： Distillation flask． E； Still head （capillary）． ． F： Exchange adapter． G： Adapter． T1， T2， T3：Ther皿ometer．

constants to be the mixtures of two adjacent esters respectively． The percentages of each definite carbon number of the ester， calculated from the saponification values and the iodine values， were 16 per cent of Cn acid esters， 34 per cent of C，， acid esters， 35 per cent of Cis acid esters， 20 per cent of C2， acid esters and 3 per cent of C22 acid esters． Based on this calculation， it was found that the cemponent acids of “Seta− shijimi” consist chiefiy of Ci6， Ci s and 020 acids with small amounts of Ci4 and C22 acids． From 2nd fraction C，， （Fi） acid was isolated， and it was identified to be zoomaric acid．   Part 1 in alcohol solution exhibited an ultra− violet absorption as shown in Fig． 8， where the characteristic absorption maxima appeared at the wave lengths of 272， 283 and 294 my． Since this spectrum sugge＄ted the presence ’of a A5，’ sterol， the unsaponifiable matter in Part 1 was exami ned， and the sterol was estimated to be corbisterol．29）        h      ／       Table 9． Remaining amount of NH2一   D） The acetone−insoluble lipids： the lecithin． the cephalins and the cerebroside．   Part II， from its constants as shown in Table 7， seemed to confain much of phospholipids． The phospholipids are， in general， separated into two groups， lecithin and cephalin， by the solubility of alcohol．20） The former is soluble， and the latter insoluble in alcohol． The part， therefQre， was separated by the treatment with 95 per cent alcohol into the alcohol−soluble lipids （53．7 ％）， Part Ila， and the insoluble ones （46．3 ％）， Part Ilb． The former was supposed to be lecithin and the latter cephalin．  i） The lecithin．一 The crude lecithin， in order to purify， were converted into their cadmium chloride salts by a usual manner， and the white double salts obtained were washed many times with dry ether until the salts became free from the compound having amino−N as an impurity．3i）  Tlie materials obtained by washing of 23 times， N （per cent） determined by Van Slyke’s apparatus．

Number of times  Salnple  N2（gas）浮唐?пimg．）    （cc．）      監

Temp．  Press．

i℃）   （皿mHg）        l         l

NH2−N

i％）

400

12

26．4 38．3 O．10 0．03

7811

755 759 O．22 0．04 as will be seen fro】：n the above table， scarecely contained the amino compound， and amounts of total nitrogen and phosphorus of them were deter− mined to the 1．43 per cent and 3，01 per cent re．       ド spectively． As the molar ratio， NIP， computed by the above values becomes 1．05， the materials seemed to． be considerably pure cadmium salts of lecithin．   The salts thus．prepared were hydrolyzed with 2．per cent barium hydroxide solution， and from the hydroiyzates the author insolated unsaturated and saturated fatty acids（palmitic acid was comfirmed），glycerophospholic acid and the cho1辻1e as its chloroplatinate．   From these results， the author concluded the presence of Iecithin in the‘‘Setashijimi”1ipids．   ii） The cephalins．一 Prior to describ三ngon the nlussel cephalins， the author roughly エnentions o且the recent study of cephalins ．obtained from other tissues． Thudichum32）was the first to dif一 ferentiate cephalin from lecithin by separation of the two phospholipids in alcohol， Bauman33） stated that cephalin obtained from an ox brain， on hydrolysis， yields a fatty acid mixture which consists of saturated and unsaturated acids， gly− cerophosphoric acid and ethanolamine inthe hydro− lyzates． Since then the difference between lecithin and cephalin had been long believed to be that the former has cholin while the latter ethanol− amine， as a nitrogenous constituent． Recently， however， Folch demonstrated that the compound formerly described as “cephalin” is actually a mixture ef several components， two of which have been identified to be phosphatidyl ethanol− amine and phosphatidyl serine， and by the subs− equent discovery that the cephalin fractions from soybean， egg yolk and brain contain inositol． ，Moreover the discovery that attracts our great interests has been the significant fact that there exist arabinose and galactose3 i） in the above

セタシジミの生化学的研究

cephalins． ln studying such cephalins， the many methods（）f p頗fying the皿have been deviced， such as the method of chro皿atogram with mag且e− sium salts，35） cadmium chloride，36） and lead ace− tate37） or that of using organic solvents，3S） but from the experimental results none of these 皿ethods is said to be su伍cient．   As known from the above mentions， the study of cephalin has been mainly directed to that of the higher animals and plants， and nQbody has studied on the cephalin ef mussels which contain 量tso 皿uch．   The crude cephalin， Part Ilb， obtained fro皿 “ Setashijimi ” was a dark brown viscous liquids having iodine value 111．5， P 2．56 ％ and N 1．85 ％． ln order to examine firstly nitrogenous co皿ponents of this cephalin， a portion of it was hydrolyzed with 6N−hydrochloric acid， and the hydrolyzates produced were filtered． The liquor 創tered was evaporated to dryness under reducing pressure， and then demonstrated by a paper− chromatography． The results were as follows ：   For the purpose of isolating a pure cephalin from the crude one， the method diviced by Folch， using chloroform and alcohol as solvents， was e皿ployed， and the white amorphous materials obtained at 5th step were examined， characteris− tics of which showed the following： M． P． 2100C （decomposing）， P 5．09 ％ （containing 1．12 ％ of inorg． P）， N． 1．88％， iodine value 85．5 （Wijs）， ash 18．6 ％ and yield 8．74 ％ （to the total amount of the crude cephalin）． They exhjbited the absence of sugar with anthrone and that of protein by BiureVs reaction． After hydrolyzing them with 6N−hydrochloric acid， the paperchromatography of the demonstration of amino compounds indi− cated that there are serine and ethanolamine （very small） in the hydro1yzates （Table 11）． Table 11．

Spot

Solvent

Serine

Ethanolamine

Butanol ： Acetic acid ：   Water （4：2： 1）  O．28 （O．44）ecee

Phenol

 O．35 （O．10）acee Table 10． Rf va】ues of nitrogenous co皿Ponents・

譜聖

Unknown

Unknown

Serine Glutamic acid

Ethanolamine

Butanol： Acetic acid：   Water （4 ： 2 ： 1）

Phenol

O．09 0．15 0．29 Q．34 0．45 e．ol O．45 0．34 0．21 0．09   From the paperchromatography of nitrogenous coエnponents，（Table 10）， serine， ethanolam圭ne， glutarnic acidee and other unknown amino com− pounds were found in the hydrolyzates． The ce− phalin， therefore， was supposed to be a皿ixture of the components containing various klnds of base． But the cephalin showed the absence of inositol by Salkowski’s method3r）） and that of reducing sugar with Fehling solution， from which it was found to be different from the cephalin of a soybean， an egg and an ox brain．   Accordingly， the white materials seerried to be a comparatively pure serine cephalin， and etha− nolamine cephalin and the compounds containing glutamic acid and other amino compounds were re− mained in the soluble part by the Folch procedure．   In order to confirm the皿aterials to be serine cephalin， they were hydrolyzed with acid and alkali， and their hydrolyzates were examined by a usual manner． Stearic acid， palmitic acid， oleic acid and glycerophosphoric acids were iso− lated and confirmed． ln addition， the nitrogenous base in the hydrolyzate was proved to be serine as p−hydroxyazobenzene−p’一sulfonate． From these results， one of the cephalins prepared from “ Setashijimi ” was supposed to contain pqlmityl− oleyl−phosphatidyl serine and stearyl−oleyl−phos− phatidyl serine． From the existence of ethanol− amine（Table 11） in the initial hydrolyzates “ Seta− shijimi” is supposed to contain ethanolamine ee The Rf value of glutamic acid （and of the standard acid） by the further paperchromatographic demonstration was O．20 （O．21） in phenol， O．21 （O．20） in butanol−acetic acid and O．09 （O．08） in lutidine− eollidine． This was presented at the 8th annual meeting of the Chemical Society of Japan in Tokyo， April 2， 1955． ceX Very feeble spots．

eeph乙li血also， phosphaf二dyl ethanolamine， but She study・σ血the other compon6ht conta量ning glutamiな acid a．hd tw．O unk：nown nitrogenoUs．bases， is ex− P6dted三n future．40）      、 ，・     1「・ 、   iii）・The cerehroside．一41）The acetone・insQluble ！ipids gf“」SetaShilimi”（Table 7）鴨rel．（駆s箪olved i！Pether， and to the ethereal、 solution．Was added pQtroleum ether（b． p．40。∼60℃）． After coolin呂 the mixture in a．refriger興tor overnight， thq．．white alnorphous mq沁rials precipitated．．on the「bottom of the且ask∫w⑱re ’ separated off from声he liquor馬 丁皐emother liquor照s concentrated to a half of its vOlume and cooled． A small amQu郎of−the        し precipitates collected was combined with the且rst Iot and，washed．with． dry ether．．The ether・in− soluble materials， Part III， froT］4 thei耳 ProPertiqs shown in Table 7， were considered to contain a sugar−containing Iipid． In general the separation or preparation of the sugar−containing Iipid in pure state are believed to be exceeding！y difHcult         ヨ because it is insolpble in ordinary solvents， such as diethyl ether， acetone and ethyl acetate， but pyridine is said to be an excellent solvent when cooled as well as heated． TherefQre， the above r      Iト      ℃ materials were treated with pyridine by the follo・ wing Procedure to isolate them in pure state．   They、 were．pulverized， dis＄01ved in war．m pyr孟一       ロ dine， and then cooled in la refrigerator overnight．        ま       ロ

Awhite amorphous precipitate（CI）deposited

       r was centrifuged and washed with cold pyridine． To the mother Iiquor was added dry ether， after cooled as above， the precipitate （C2） deposited        I「 was centrifuged again． Moreover， to the remain− ing sohlt圭on was added dry ether， coo16d and th6n the precipitate（C3）deposited was collectedl The L’final liquOf， evaporated to dryness，1eft a residUe，（R）． The properties of these four fractions are shown．至n Table 12．    ’ Ta61e 12．  」     1

eraction ．Yieldi9）L        ，

M．P． i℃）， N． i％） P︵％︶

Ash

i％）

qG亀R

O．19 1．03 0．49 1．03 179”一一178 186・一187

187N190

  204 3．30 3．45 3．48 3．86 3．68 4．21 4．42 4．37 16．0 16．3   All of these fractions readily gave a fine suspension with water．   As seen・i血Lthe above table；the fractiohs，．C壱 and℃3， seemed to be si皿i墨ar subst2nces from their properties， so that they were m圭xedi togeth− er． The． fraction combined was recrystallized 6nce from hot methanal。 The crystals showed melting．point of、186℃， molecular weight’（Rast） 660and the presence of sugar by Molisch，s test． The analytical values of the crystalline materials were as fo110ws：C56．79，耳11．48， Total−N．3．46 （NH，一N 1．35）， P 4．31， Ash 14．02． In order to know the components， the materials were．hydr6」 1yzed with dilute sulfuric acid， and in thdr hydro一’ 1yzates fatty acids， sugar and n圭troge血ous compound ∼vere detected． Particularly that component sugar’ is demonstrat6d by a paperchromatography tσ be xylose suggest6d us a great important fact．   The propertieS such as sO玉ぬbilities， Inelting point， molecular weight and nitrogen cOntent 6xcept contents of phosphorous and ash， see皿ed． roughly cOincident with those of the cerebrosides obtained from ox bfain or another tissue． The author， therefor6’， considered that the． above’ materials separated from“Setashijimi”belong to anew class of cerebros三des等vhicL di琶er frGm， cerebrosides hitherto known because they contain㌧ xylose in place of galactose or glucose。   The crystalline materials， in oraer to re皿ove・ the impurities， such as the ash and㌻he phosphorus一㍉ containing substance， were subjected to thediaユysis by Folche’s method42）and then to the cadmium         chloride．methanol purification by Klenk’s proce− dtire．43）  Consequently the ■naterials Obtained sho．wed the following analytical vahles：C・59．68， H11，86， Total−N 2．03 （NH2−N o）， P o．10， Ash 1，28，Mol． wt．680， M。 P．1860∼187℃． From these values， these materials still contained traces of phosphorus and ash， but no further purification        b was possible．   The materials on hydrolysis yielded acids， sphingosine−1ike substances and sugar in appro− ximateiy molecular equivalent proportions． After separating th6 hydrolyzates into the． ether−soluble part（82．9 タ6） and the −insoluble one （17・1．％）， both parts were examined． From the former palmitic acidL and the sphingosine圏like substances as their sulfates were obtained and from the latter x夕lose was identified．   For the identification of the component sugar， the following test were carried out． The aqueous

セタシジミの生化学的研究

solution containing the ether−insoluble products exhibjted a positive test for sugar when reacted with anthrone and concentrated sulfuric acid， and indicated the presence of pentose when heated with orcinol （Bial’s method）． The content of reducing sugar，．determined by the皿icro一 ．Bertrand’s method， a皿ounted to 14．6 per cent of materials used for hydrolysis， and this reduc−        Table 13． Rf ing sugar was confirmed to be an aldose by Willstater−Schudel’s method． Then， in order to know what sort of pentose it was demonstrated by paperchromatography， and the following Rf values were obtained．  From this table， the sugar seems to be xylose． For the further confirmation of xylose the ab− sorption spectrum of amyl alcoholsolution of values of reducing sugars．

Spot

Solvent

Butanol： Pyridine： IWater （3：2： 1．5）

Sample

Sample十Xylose

Xylose

 Arabinose  Glucose  Galactose O．54 0．54 0．53 0．50 0．42 ’Butanol： Ethanol： Water （4：1： 5） O．52 0．52 0．52 0．50 0．48 Acetic acid ： Water： Butanol（1 ： 2 ： 4） O．23 0．23 0．24 O．14 0．12 Phenol： Water   （Saturated） O．44 0．44 0．44 0．51 the sugar reacted with orcinol was taken， which showed the characteristic maxima4r’） at 620 my and 68e mp． As seen in Fig． 9， when compared the curve of the spectrum of the sample sugar with those of xylose and arabinose， the sample was more si皿ilar to xylose than arabinose． ←刷 ﾌ信Φ℃冒り事qO   The phenylosazone prepared from the sugar was found to be identical in its crystalline form， 皿elting Point a員d nitrogen content with known xylosazone． Besides， the dibenzyiiden dimethyl aceta146） of the sugar agreed in admixed me！ting point with that of xylose．   In the hydrolyzates of the cerebroside， as above mentioned， the author confirmed the acid （palmi− tic acid） and the sugar （xylose）， while failed in isolating sphingosine in spite of his efforts． But this compound was qualitatively detected by solu一 ’bilities， melting point and nitrogen content of its sulfate derivative （Tab1e一 14）． Table 14． Co皿position of the sphingosine sulfate．        ． From calc． From “ Setashijimi       （Ci，H，，O，N，），H，SO，47） M． P．

Carbon

’Hyd’rogen ・Nitrogen Sulfuric acid Iodine value 200ce （dec．） 62．18 11．07   3．85 13．6 45．8eeee 2000C （dec．）es 61．89 10．60  4．00 14．0

     S20 700

       Wave length （my）

  一×一x一：Sample 一〇一〇一：Xylose

  −A−A一； Arabinose  Fig． 9． Absorption curves of amyl alcohol solut− ion og the reaction products by Bail’s reactlon．        幽      竃． ac Prepared’ from ox brain． eeX’ Determined by Wijs’ rnethod．  In short， the cer6broside’ separated from “ Seta− s’ ?奄鰍奄高堰hrese皿bles oth6r bne obtained from ani− mal spleen and’ bacterial lipids4g） except that it contains xylose in place of galactose of glucose， and seemed to be a new cerebroside，