[Agr. Biol. Chem., Vol. 29, No. The Part I. 10, p. 949•`955, House Extraction 1965] Fly Attractants in Mushrooms and Activities of the Attractive Components in Amanita muscaria (L) Fr. By Toshio Laboratory MUTO of . The Pesticide Tokyo and Chemistry, University Several active Amanita solvent one concentrates muscaria extraction fraction of the referred It was on long belonging to and to known fruiting the et chemical acids and acid and been rather ble to the ples the attracting by of anticipation, various agents methods adsorbent and extracting finally several with a novel of known house steam fly attracting distillation body. Among colorless crystals, exhibited a ness the exhaustive active m.p. marked property and concentrates, 22•`23•Ž, with attractiveness was isolated as to colorless chromatographycaly a house crystals proved the the with a purity. muscaria species and from the using the fruiting solvents, substances fly and established a at- fly is ex in cap in 15cm. process of September of near the the Tokyo concentrates; one comparatively strong attractive 1) T. Takemoto and M. Nakajima, Yakugaku-Zassi, 84, 1183(1964). 2) T. Takemoto and M. Nakajima, ibid., 84, 1186 (1964). 3) T. Takemoto and M. Nakajima, ibid., 84, 1230 (1964). 4) T. Takemoto and M. Nakajima, ibid., 84. 1232 (1964). bodies were off-white the in Life a left of parts the up the from pink 10•` to about mushroom the at Research the warts, stalk October University end the of region Institute of of Education in Prefecture. harvest, within as mushroom by period early Nagano After This bodies a Plateau Sugadaira, lost and in known used mountaneous with fruiting to mentioned the and northern and Amanita above characterized harvested a same easily extraction. spotted The precipition the property the using possessing of of selected from diameter, were active Fr. and colored of obtainable mushrooms Japan Preservation advantages attracting native and authors material 15cm body ion house of With extracted (L) of RESULTS Potency mushroom the starting princi mushrooms. Mushroom Attracting abundantly, attracted AND the of remarka house the Fly Considering had mushrooms active of House species we marked have the amino are some a Harvest toxic tricholomic another flies the EXPERIMENTAL death. acidic of the of such we components changers, them bodies potency tractive in house for and elucidated However, that existence responsible this as acid.1•`4) fruiting supposed isolated names interested as gather to mushrooms, the phenomenon long Tricholomataceae structures ibotenic the fruiting mushrooms poisoned successfully proposed the isolated flies some get those of 1965 through homogeneity house families al. in was 9, Agriculture Tokyo females. of and components their that for obtained juice D3 mature bodies Amanitaceae Takemoto as proved especially the were filtered to chromatographycally flies, Fr. of Education, June responsible (L.) SUGAWARA Faculty of Received of Ryozo the few at attracting weeks ambient potency when the conditions; was fruiting even 950 Toshio preserved in a refrigerator disappeared a within process consisting bodies a cloth and placing found in to be the original loss of six covering a refrigerator quite the was storage. stored was detected Hence used for tion pH of 0•Ž no of extraction of room width, controlled in at at along the Three 26•‹ cedure, at to side a height hundreds of after one and L.), left Petri more dishes, were 2cm 20 placed dishes an to 30ml of the shelf the the in the usually while supply the with centre potencies pared had that flies for of based fallen that number of (16 hours) samples on of of and the visited the visiting be placed the drowned flies the in which drowned during number of the far ex basins flies, counted both over to about in to 7. distilled the distilled procedure to was distillate 500ml bath the pH intermittently added to adjusted the to original repeated exhibited to concentration, was was a The water while After above debris. a being water solution as through concentrated flask done ranges: prepared filtered in pH 10. stable 60•`70•Ž the at was some was the within above and pressure back again and remove filtrate solution of 30min. juice 7 at decreases specified pH 12 condition, work filtered to at and the the does reflux beyond 2 the juice 1 to the markedly reduced and in proved filtered extraction Celite flask ap Components from below the brown This com flies of 500ml attractive mutually layer adjusted water and the test the retained but pH of the Active under in adjusted under after diluted obtaining ranging the was of illumina a 9, maintained time, and The counts Although the may be were of is to at dark to dishes under milk basins variable period were water, samples Petri room. the the but the cotton the having ceeded suitable powdered into really a the period. 30cm of of of in pH potency 3000ml with volume than wicked solutions the and enrichment stability throughout diameter, test at Hence alternatively The overnight tion, the room in aqueous number simultaneously. experiment. example, step. of temperature; 7 pro mutual Juice. drop 10min. room that potency from test suitably of for in preliminary not the days the Filtered room (Musca 4 in 9cm same longer to so Five some before and on interval tested left deep containing according than longer shelf flies released yield Fractionation potency this for grade tested accurate before were the Primary A potencies, extraction, to attracting were shelf. of simplicity an require concentrates proximate entrance. the the step of with latter extracting attracting that their the the Since samples of concentra of reasons the flies time simultaneously guide impossible or according the floor each, house were or the from over of the watts 53cm aged 30min. containing from thousand emergence, width from 20 evenly to domestica wall a a the of simultaneously height, humidity, with 62cm lights, in relative shelf of illuminate 245cm in by expression evaluation a 325cm as reproducibility. the the in the of Therefore, employed considered of period with components components good juice Potency of 60% a height left Attracting and and with a Fly dimensions depth fluoresent fixed as House the equipped 30cm at of with 294cm was active and number longer experiments. solutions Procedure A the a correlation active was was active and after good performed after principles. Assay the unitary apparent filtered a of count was even the of time drowned showed retention potency; potency period found toluene about SUGAWARA fruiting the for Ryozo filtering with at effective attracting months' thus adjusting 7•`8, potency the at and However, harvest, cotton, juice the crushing after of filtered -2•Ž, months. of immediately with the at three MUTO twice no the pH volume. more until detectable attrac tiveness. The distilled extracted the The a with with organic water, portions were 400ml hexane layers dried were over combined combined, anhydrous and three times. washed Na2SO4 and 7 The House Fly Attractants distilled. The residual pale yellow oil was dissolved in a small quantity of hexane and charged on a column of alumina prepared by pouring a hexane slurry of 50g material into a glass tube, 4cm in diameter. The column was developed with hexane followed by methanol. The hexane eluate (300ml) and methanol eluate (100ml) on evaporation of the solvents yielded 440mg of colorless oil and 150mg of yellowish viscous liquid. Both residues exhibited house fly attracting potencies when tested with the above specified pro cedure. Thus they were referred to as the fractions A and B respectively. The residual solution after the above steam distillations still retained a remarkable house fly attracting potency. After a variety of solvents at several pH conditions had been examined regarding the efficiency of extracting the residual components, nearly all the re maining potency was found to be removed by exhaustive extraction with chloroform followed by butanol at pH 2. Thus the residual solu tion, 500ml in volume, was adjusted to pH 2 and repeatedly shaken with 50ml portion of chloroform. The chloroform layers were separated, combined (ca. 500ml), washed with water, dried over Na2SO4 and evaporated under reduced pressure. The viscous brown residue was dissolved in a small volume of hexane and charged to a column prepared by pouring a hexane slurry of 50g alumina into a glass tube, 4cm in diameter, and allowing to settle. The column was eluted in succes sion with 500ml of hexane, 500ml of benzene, 1000ml of 1% methanol-benzene, 500ml of 5% methanol-benzene and finally 100ml of methanol. The hexane eluate on evaporation of the solvent yielded 75mg of colorless vis cous oil; 1% methanol-benzene eluate, 946mg of pale yellow viscous residue; and 5% methanol-benzene eluate, 113mg of pale yellow viscous residue. These concentrates which exhibited house fly attracting potencies in the above described methods were referred to as C, D and E respectively. Some residual in Mushrooms. potency Part I remaining extraction in with removed with the butanol; to the after was the filtrate house after the completely flies the became extraction butanol. Thin-Layer Chromatography Homogeneity Since of the the primarily a an the thin-layer and to Silica Gel carrier. adsorbing ones, of was water paste and applied over thickness of 0.25mm. by and samples heating lengths in an oven, were a glass 105•Ž 9 to 11cm with in oven with for with 2% acetate-benzene a glass 20% PHOTO. 1. Thin-Layer Chromatograms D and Crystalline D3. The left plate was the right plate, with being spotted from two crystallizations an to were spotted either in with plates plates plates ethyl an the homogeneous The 10% as of mixed to developed sprayed Co. grams clean The or from Merck throughly at cooled. methanol-benzene to employing G. material further checked Kagakukikai Twenty-five distilled most among was being Yamato of 30min. flies it by 50ml activated the the house homogeneity made adsorbent test exhibited chromatography apparatus Co. D separated while Checking D. attractiveness purified with for Fraction fraction remarkable a filtrate chloroform with indifferent 951 jar, sulfuric dried acid of a Crude developed with 2% methanol-benzene; 10% ethyl acetate-benzene, each plate left to right with crude D, D3 after and D3 after three crystallizations. 952 Toshio and again shown heated in typically spot brown spots the three of of 0.14•`0.16, the and D3, 0.48•`0.53 in The from RF values in 2% Purification While the checked of with purified the gel (a into allowed The crude a a 1% methanol-benzene rated of and The spot D3 of the D1 and the solvent, only, D2 spots. eluate and and 5ml more D3, slightly six FIG. 1. by those 50ml the the frac fractions giving fractions of end the solution substance; spectrum 75.51%; in 2920, 2848, 1655, 1460, 1413, 1378, LR. Spectrum of Crystalline D3 (liquid film). in ethyl less hexane, and characteristic ultraviolet region measured in concentration 1mg/ml absorption measured 3000, a 0, somewhat when the for (1.79% no the absorption a 11.54%; 11.31%; and water; 3480, H, chloroform, ether, at molecu method, [ƒ¿]D=•}0 in the white H, ethanol maximum an by apparent the Rast's 75.56%; and in flies, obtained calculated solube methanol, hexane infrared C, 731.12; soluble except a an with C, benzene in absorption about W. very acetate, benzene of the M. house far properties: difference), 13.13%; soluble so characterized analysis, C46H820b: exhibited a chromatographically was (by chloroform); in Crystal to fraction with formula, evapo than passage finally The cut was in followed D2 to chromatography. nothing of D2 the dissolved after elementary with the separated potencies a m.p. 22•`23•Ž; determined 12.95% charged colorless which physico-chemical 0, developed fraction thin-layer giving consisting containing both residue and was following 693; dissolved attractive crystals diameter, 12cm. of concentrates D3, of of was benzene and appeared in of procedure homogeneity, platelets; lar weight benzene, height (670mg) Each to the fraction 1000ml tions the a column 50ml. subjected 4cm to of The fractions and tube, settle volume column. Chemi in several less form proved 50g crystallizations 313mg Properties above component in further Mallinckrodt suspended fraction the was repeated and residue. 22•`23•Ž. the the or the thin-layer D chromatography; of glass D small gel was to described being combined, waxy D3 more eluates on yield m.p. Among D of fraction product Co.) and in silica Works Fraction above the with silica the only were a colorless Physico-Chemical line acetate-benzene, homogeneity chromatography, poured Some 0.30•`0.33 spot give ether-methanol through Further cal waxy platelets, respectively. of residue coloring 0.55•`0.61 ethyl D3 to coloring the SUGAWARA giving the evaporated rapidly 0.08•`0.10, 10% As concentrate Ryozo slowly with and and and min. D the and D2 0.37•`0.43 for30 crude spots: D1, methanol-benzene and 115•Ž 1, gave violet of at Photo. MUTO maxima as 1743, 1238, liquid 1720 1167, in a film at (shoulder), 1118, 1091, The House FIG. 2. 1017, 940 NMR and 721cm-1 spectrum was measured thyl in silane House as Fly CCl4 an NMR Spectrum a 60mc. including 2 whitish with which in Potencies of water filtered Amanita juice muscaria tracted active less attracting flies gathered active of (L.) the Fr. juice, solutions tained during crystalline be Ex. of of distilled the distance about emergence released. 500 of ten Within To were 60cm from days one containing on a trace the a batch 5 hours, had 159 which on Petri-dish, placed room in having been flies a which at previously females major the started in a to the flies as the in crystalline a hexane 60cm the the solution the were After females the flies males) was food. gradu 24 again 210 a dish, from food basin. of and on 15 cm a in of (452 an shelf. 22•`23•Ž) giving chromatography over the well in the and of the as gathered water proportion abdomen on deep dropped distance and Then milk had 12 room wicked 30mg swarming drowned Ex. started (138 at whitish in flies 3cm distilled attracted 662 30ml been of flies ally the out hatch hours. cotton of D3 (m.p. on the thin-layer 100ml The shelf the taken the placed containing had thin-layer the only in to in 10 of dish, flies powdered were all Petri preparation single spot This died died started for supply nearly a (m.p. 20mg even while house with flask D3 hexane. carrying and males) male) released filled water diameter, dropped one feeding examples containing placed in before a 21 sample, had were dishes a food, flies eggs and test which Erlenmyer After the with thousand batch without ob diameter, 1ml left and with was before as the and One days extraction the 2. a Petri original of of female from concentrates by in another water flies the dropped the several used. in dissolved and of distilled them of water sample Petri-dish flies; Of revealed was 30ml of contaminated 9cm and or containing preparation as deep a fraction, ex below. chromatography of D3 D, 2cm the of basin. Ex. of more house and course still amount as dishes some the Crystalline 20•`21•Ž) to drowned. described 1. well Petri and filtered bodies exhibited the and will as potencies to basins the fruiting concentrates concentrates into the portion ovipositor basin (one 953 swollen projecting two Extracted a major the I D3 (CC14). abdomen those tetrame Part of Crystalline and, Components The Mushrooms. Fig. standard. Attracting in 1): solution internal Attractants in (Fig. represented Fly hours, including with swollen, were found basin. 3. About 500 emergence room. 5 The flies from days before Petri a batch dishes were having released containing 954 4mg, Toshio 2mg and 1mg of the same MUTO and crystalline Ryozo SUGAWARA Amanitaceae, preparation of D3 dropped to 30ml water were placed alternatively with three Petri dishes containing distilled water at an interval of 40cm. After 16 hours, 26 (18 females and 8 males), 22 (16 females and 6 males) and 12 aiming (8 females and 4 males) flies died in the dishes containing 4mg, 2mg and 1mg of the sample, respectively, while 3 (1 male and 2 females), 6 (1 male and 5 females) and 3 (4 females) died in the water basins. After 10 days of the above experiment, 500 flies from the same batch, thus having passed 15 days after the commencement of emergence were released in the room and the house fly attracting potency of the same sample was similarly tested. Within 16 hours 27 (25 females and 2 males), 11 (7 females and 4 males) and 12 (4 females and 8 males) flies drowned in the basins containing 300, 30 and 3 mcg per dish respectively, while only one fly was found in each of the three water basins. A comparison of these two similar experi ments carried out at the interval of 10 days as well as other experiments suggested the sensitivity of house flies to D3 increases along with the adult days after emergence. Ex. 4. A sample of A, somewhat purified by a vacuum distillation but not yet suf ficiently pure for establishing a strict identity, was added to 30ml of distilled water in Petri dishes, 9cm in diameter, at rates of 10mg, 1mg and 0.1mg per dish. The Petri dishes were placed on the shelf alternatively with those containing distilled water. After 16 hours, the numbers of drowned flies were 111, 27 and 9 in the basins of 10mg, 1mg, and 0.1mg respectively, while the numbers of the drowned flies in the water basins were 9, 9 and 2. period DISCUSSION Anticipating any principles the long known phenomenon for flies gather to the fruiting bodies of the mushrooms belonging to the families Tricholomaceae and the ponents. of material a essentially bodies obtainable in liquid and after satisfactory Of the ated for retaining active through the exhaustive referred to as of I. R. and istence straight and free fatty well suggested a the of ex a long, carbonyl method neither were group rather times employed so accurate in nor satisfied results many the weight, residue test reproduced peated form problem, molecular acid, is we in structure group. the experiment and component, isolated another esterified hydroxyl sensitive, fraction chemical spectra an Admitting this is chain a be analysis, NMR of be quite distillation a the elementary im to potency. of could component of fruiting portions found the Although this data process the extraction, D3, crystals. at limited concentrates steps solvent very solid was several a disclosed separating harvest first fly a valuable in above consisting into mediately house mushrooms, the com were the the year, experiments supposed difficulties preserving potency of the some in starting out of Although tracting of carried isolation experienced so with the of similar tests during the course re of extraction. Seemingly, D3 and the other with advance some 4•`5 increased to or of for in drowned the the the of increasing more, the large drowned and proportion showing of increased might the those at high suggested which flies males; even found it was flies the those than females were regard, of the the sensitive house maturity and sensitivity in abdomen to appeared particularly more flies, sensitivity with Still ovipositors In this it apparent females swollen projecting frequency. to food. were flies increased emergence females, eggs house life; their their the suggested bearing whitish, adult after with of of concentrates their along desire flies sensitivity active days proportion the responsible that house we at egg be due bearing females. The results of this experiment clearly proved The House Fly Attractants the long known ability of the mushroom to attract house flies is attributable to some active components present in the fruiting body. Since the fractions other than D3, for example, A, C, D2 and E, though minor components, are supposed to have not so inferior levels of activity and relations with D, in structure, we now intend further purification of these frac tions as well as the elucidation of the chemical structure of D3. in Mushrooms. Part I Acknowledgement. Dr. H. Ando of Science, for for their Mr. Tokyo kindest harvesting was of K. are very Sato the of University instructions the investigation grant-in-aid We and the 955 Ministry to Faculty of Education and mushrooms. partly grateful the Also, suppoted of cooperation by Education. this the