THE INVESTBGATSON OF CERTAIN ANTIBACTERIAL!) lN POPULUS TACAMAHACA MILL. AND HYPERSCUM PROLIFICUM W.“ Thesis éor the Dem of Ph. D. MICHQMN STATE UNIVERSITY Gamid Gean Duil 1956 THESIS . H '/ . \ 5., z.‘_ — “~A --_ w L [B R f1 .0"? ‘1" Michigan 3 ra [c Univrrsity —‘ "cw-Ir """ {NW 1 3 ' .r ‘ T}; :‘j 5,2 l *5: a; J, ms WOMEN OF CERTAIN ANTIBACTERIALS IN POZULUS TACMAHAQA HELL. AND HIPEQICUM PROLIFICUM mmm A THESIS Submitted to the School of Advanced amt. Studio: of Michigan State University of Agriculture and Applied Science in penal fulfillment. of the roqnirmu for the dogma of DOCTOR OF PHILOSOPEI Deputment of Chemistry 1956 /r'/:I‘-3I t, 7530 ACKNOWC‘E'ERHS The author wishes to express his gratitude to Dr. James L. Fairley for the encouragement end stimu- hting guidance throughout. the duration of this work. The author would also like to thank Dr. E. H. Lucas, who initiated this problem, for his help and sug- potions; The author would also like to express his thanks to the National Institute of Health for providing funds in support of this work. W The euthor was born in Laurel, Montana on the 20th of August, 1930. He received his secondary schooling at Glasgow nigh School, Glasgov, Montana. The author graduated from Montana state College in Boseman, Mans, June 1952 with a Bachelor of Science degee in chemis- try. He enrolled at Michigan State University in September 1952. During his time at Michigan State University, he was a Graduate Teaching Assistant for eight quarters and a Special Graduate Research Assistant for seven quarters. He also worked for the E. I. duPont deflmurs Com during the sumners o! The author is a member of sigma Xi and Phi Kappa Phi. me INVESTIGATION OF (1me ANTIBACTERIAIS IN POPULUB TACAEAHLCL HILL. AND PEPFZEICUM PROLIFICUIIE Gerald GeanDull Submitted to the School of Advanced Graduate Studies of hichigen state University or Agriculmre and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR 0! £231.03er Departnmt or Chemistry Tear 1956 W 6, i- W l. V i LBSYRACT TI: seequiterpenc alcohols, one or which was identified as an iscneretbisebolol, vereisolatedrronthebudsormucmhsca Hill. by neans o! solvent extraction, Maul distillation and chip-stopaphic techniques. Both substances showed in m activity spilt mafia-inn tuberculosis at the level of five micrograms per silliliter. in ethyl acetate «tract of the flowers or g. mliflcxm yielded, after purification by adsorption chronetOgraphy, a highly antibacterial We. The structure of this principle has not been established as mutthesubstancehasbeenmcmtocontadnanaremtic moleus,a Wmandacaxbowlmp. Thsabmeotnitrogen, sulfur sndhalogeneheebeeneatabliehedanditappeerethatthemleculeis ewes-detenlyearbon, Wmmmo TABLE OF CONTENTS Page WIOEOOCOOOOCOOOOOOOOOOIOOOOUOOOCC.COOOOOOOUIOOOOOOOOOOICOOIO 1 PART I mmon or some mmc'rmm. PRINCIPLES IN ems or POPULUS game; m. macrIOHOOOOOOO00.0.0.0...QOOOQOOCOOOOOIOOOO.ICO00.000.000.00. mmmmn AND RFsmITSO00......OOOOOOOOIOOCOOQOC.OO‘IO0.0.0.0... Bio“"¥eeeeeeeeeeeeeeeeesseeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Bource 0: Crude Material.....a.s.....oo....o..............o.o PGCaration 0f orUdO Extract.........oo..a.a.e........o.a.... ‘1k311n9 EXtractioneeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee ‘Oidic EItrICt1°neeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeoeeeeeeeeee Simple Diltillationeeeeeeeeeeeeeeeeeeeeeeeeoeeeeoeeeeeeeeeeee Frictional Diltillfltioneeeeeeeeeeee.eeseeeeeeeeeeeeeeeeeeeeee Chromatographic Purificationeeeeeeeeeeeeeeseeeeeeeeeeeeeeeeee Cb-chChthfl\fiUfl U1 h: N G) wasIONOOOOQOOOOUO0.00....COOOUIOODOOCOCICIOOOOOO...OOOOOOOIOC. PART II INVESTIGATION OF ANTIBACTERIAL PROPERTIB OF THE FLOWER OF HIPERICUM PROLIFICUH INTRODUCHONOCOIOOOQQOQOOOOOOOOQOOIO.IOIOOOOOCGOOQOICOOIOCQOIIO... 3h mmn m RESULTSeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 36 31“”.0.0.00.0...00.00.000.00...IOOOOOOOOOOOIOOOIOOOOCO... 36 Source 0: Crude Hhfiflrilleeeeoeeeeeeeeeeeeeeeeeeseeeeeeeeeeeee 36 Preparation.cf thO Crude EXtPIOteeeeeeeeeeeeeeeeeeeeeeeaoeeee 36 ’rsperation.of Stock Selution........o...............e....... 38 ‘01d10 and Alkaline Extraction.of the-Stock Solutionm..s.o... 39 Belection Of Adsorbent......................3....e.a.oo...... bl Chromatographic Purif1¢.ti°nThy Elntioneeeeeeeeeeeeeeeeeeeee h3 Chromatogrsphic Puriticationflhy Extrusion.................... “6 vi TAILS OF CONTENTS . Continued Page stlitative Intonation concerning Some Active Preparations. 51 Stability in Heat and ‘1reeeeeeeeeeeeeeeeeeeeeeeeeeeeee 51 Bt‘bility in soxvantfleeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 51 Elemental.Analysis..................................... 5h Dmxou...’.00....0.OOOOOCOOOOOOCOQC.‘OII....OOOOOOIO0.0.0.... 55 WIOOOOQOOIOOOOOOOOCIOOOOOOOOOOIOQOOOOOCOOQOOOOOOOCOOO090...! 57 mmm...OOOCOOCCOOOIOOIOOOOOOOOOOOCOOOOIOOOOCOOOIIOOOOOOO. 58 mmxcmOIOOOQO0.0.0....00.00....IOOIOCOOOOOOQOOOOOO0.0.00.0... I Chromatographic Techniqual..oe........................... Column.Preparation.....................o........... Addition 0f SamPIO.................5............... Gradient Bluticfleeee .............d.6..a........... Fraction Collection and Treatment.................. II 31010813‘1 ‘Bti'1t7eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Bioalsay............o.............................. Activity Expression.and.Calculationaooo.........5.. Interpretation.of Bioassay'Results................. Total AOtiVitreeeeeeeeeeeeeeeeseeeeeeeeeeeeeeeeeeee 8281?? 83 $23838 8‘ 8‘ Ch .¢ m Hat'arid.00000.0.00.01.00.00.9.00... .QOIOOOOOOOOOOOOOOOO 55‘ scl'ent'OOOO0.0.00.00.00.00.00QCOOCOOIOOOO0.0.0.... 0‘ .g Adsofbcntl...........................5...........a. Other AdBOfbentleeeeeeeeeeeaeeeeeeeeeeeeeeeeeeeeeee 0‘ CD E HaganggannuaaflveQBnr-o LIST OF TABLES Page Biological Activities of Fractions Tron Preliminary ,TOOCdHIOIeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Data for Fractionation of neutral Oil.................... Chromatographic Data for Hediun Boiling Fraction......... Ghronatop‘aphic Data for 3.129........................... Physical Properties of D-138............................. Correlation of Activity and Oxygen Content of 13-138.... .. Chromatographic Data for D-123........................... Chromatographic Data for High Boiling Fraction........... ChromatOgraphic Data tarnish Boiling Fraction........... mum Properties and Activities of 13.210, 211, 212.... Activity of Several Tex-panes Against Bactu-ia............ Plvsical Properties of 13-212 and Bisabolol............... Results of Various Extraction Procedures Iith Ibpericun ’1."r.eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Bicassay Data on Acidic and Alkaline Extraction of Stock _Solution.Using 0330003 .fld.NH‘OHeeeeeeeeeeeeeeeeeeeeeeeee Bioassq Data on Acidic and Alkaline Extraction of Stock 8013$19D.UI1D8 501 IndtKoneeeeeeeeeeeeeeeeeeeeeeeeseeeeee Adserbents which Retain-Activity tron Hypericun Stock selntionseeeeeeeeeeeeeeeeeeeeeeeeoeeeeeeeeeeeeeeeeeeeeeee Adsorbents which Us Hot Retain Activity tron Wm “TOOK Ioluticn........................................... Data for Elution Chromatogran Tron pH 2 Alunina.......... D.“ for Elation Climate” PM pH 1‘ Waco-sees... ESKGKfiSm-a LIST OF MES «- Continued Page Date for Extrusion Chrontop‘am From pH 2 Alumina......... 149 Data to:- Uhroutog-an ct n-hSO............................ 50 Data tor Chum-atom or 341119."......................... 50 Antibacterial 'Aofluties'of Crude Extracts After Long' Standing...noun-nu...".............................. Sh Egfing LIST OF FIGURES FIGURE Page 1. Ultrsviolet spectrum of 13-138............................ 2. Infrared Spectrun of D-138............................... 3. Infrared Spectrum of D'Weeeeeeeeeeeeeeeseeeeeeeeeeseeee h. Ultraviolet Spectrum of mm...” 5. Infrared Spectra: of High Boiling Fraction............... 6. Innared Bpeotrun of mu"........................... 7. Infrared spectrum of D-le............................... 8. Infrared Spectrum of D-le............................... 9. Infrared Spectrum of n—m............................... l0. Infrared spectra: ei’ Wu.......................... 11. Ultrsviolst Spectrum of H482...”....................... 12. Infrared Spam of 3-282............................... 13. Ultraviolet Spectra: of 3462......”uu............... 1h. Ultraviolet lpectm or 3466............................ 13 1h 17 18 20 23 2h 25 31 32 h? In 52 53 HISTORICAL Iran the start of recorded history man has been engaged in a fight against infectious diseases, and the plant sorld has provided a bounti- on source of materials used in this struggle. Ancient Chinese literature records that alga-w (2738—2698 B. 0.) investigated the phsnscological effect of some plant materials. In the new centuries follssing his sork additional information was collected and in 1597 A. n. 1.; Mg produced a SZ-wolune series on Chinese medicinal nterials (including many plant sources) entitled ran-woo hang-nut. An intu'esting discussion of Chinese medicinal naterials has been pre- pared by hosig and Cchram (1)., The Chinese sore not alone in their endeavors to find plant sources of medicines, as the Baptisms were also ulightened in this area. The Papyrus were (1500 B. C.) (2) represents another sample of ancient records finish include descriptions of the useefplante for medicinalpurposes. Theartofusingplants as sources of sedicaticne for mean ailnents sas kept alive by Hippocrates (1.60- 361 s. c) and Galen (103-193 a. 12.). These no sen helped bridge the gap fromthetime ofthe Chinese herbists to the seventeenth century herbistssuohaslohnaerard. Gerard, ashalisluan, miledalarge assent ef intonation on herbs. This intonation new be found in the revised edition of The am; of cm gators... of Tlants (3). Cersrd reported that an extract of the cyperus plant could affect ,nraoulouecuresofillnesees rangingfrondropsyandsnakebitesto Intel tension. Although such of the early ion: on done with crude plant contracts, sore recent chemotherapeutic imaetigations concern the isolation and idutdfioation of specific compounds from both high and los ferns of plants. The isolation of penicillin (14) from Penicillium m is a classic example of a loser plant being the source of a chemotherapeutic W. lisilar isolations have been done with higher plants but results have bear sonemt lees spectacular. The red and yellow varieties of the canon onion ($132325) are not susceptible to the fungal disease Colletomchun circinans (Berk) chl, while the uhite varieties are susceptible. In 1933 Link 31 53,. (S) isolated 3,5-dihydroxybensoie acid (protocatechuic acid) from the scales of the pigmented variety. The compoundusfcundtobe toxictog. circinansinanaqueousdiluticn of 1.3000. rmm purification of extracts of sweet potato (6), banana (7). and Indian carrot (8) has produced materials with weak antibacterisl activity. Lucas and comrkers have been conducting an utensive screening program (9) sith the intent of finding seed plant sources of potent antibacterials. They found (10) that extracts of easy plants desenstrated appreciable antibacterial activity. Of these, nan-emu. «Wanner «we «and. estrasly high antibacterial activities. 0n the basis of these findings this research project was initiated sith the purpose of isolating and identifying the sain active principles of these tee plants. PART I INVESTIGATION OF SOME ANTIBACTERIAL PRINCIPLES IN BUDS OF POPULUS TACAMAHACA.HILL. INTRODWTION A comercially available oil (Balm of Gilead) from the buds of m candicans lit. and 22221.25. tacemehaoa Mill. (also known as m balsamifers 1..) has been used in the perfume industry for new years. However, the first interest in the antibacterial properties of poplar bud oil arose when Lucas and occuorkers found (10) that ethanolic and bet aqueous extracts of the leaves and buds of {. tacoxmhaca were active against Embacterium tuberculosis and morococcwus; m, var. m. On the basis of the findings of Lucas 33; Q..." fillies! Bonff and George Levitt carried out some pralindnary isolation uperinents (unpublished york) under the guidance of Dr. H. H. Bell. These experi- tents were unsuccessml with respect to the isolation of, the active principle involved. Prior to the interest in the antibacterial properties, several groups did considerable nor]: on the isolation and identification of constituents of peplar bud oil. In 19224, Nekao (ll) isolated the sesquiterpene 'populene' from the buds of mm. He also men- tioned the possibility of the presence of sesquiterpene alcohols in the oil. In 1936, Corie and Canal (12) isolated and identified mam constituents of poplar bud oil from I.- balsenifera, one of Ihich was a mean-rpm doohol. tom 33 a}... (13), in 1952, identified the aner terpuxic constituents in poplar bed oil from ;. balsaniferg as cinecl, daeadinene, ar-cnrcnnen, farnesene and cc-dobisabclol. Using this previous ark as a basis, the isolation and identification of the antibacterial substances in poplar bud oil front. tecanahaca wee usertaken. EDEBDIEHTH. AND RESULTS 24.0.2151 The purification procedures were followed by biological assay. against muctuiun tuberculosis and Microccccus we; var. m. Il'he sews were designed and directed by Dr. R. I. Gottehall at the Division of Laboratories, Michigan Department of Health. A discussion of the methods of assay sill be found in Appendix II. the activities are expressed as the least umber of aicroyans ( 8) per nilliliter of culture broth uhich inhibits bacterial grevth. wofcrudefieterial Thepcplarbudsueedinthisresearchvsreobtainedfrcntw senses. inixtureofbudeofg. tscamhacsandz. sendicguspro- eurellfrenthsl. I. Penick Conpalv. Budsfronz.‘ tacsnahecaeerenadc available tln'eugh the ccurtesyef Professor )1. If. new, superintendent of the Mar Forest W Station of Hichigan state University at Isllte Its. Harie, Michigan. mum of Crude Extra Afifteenkilogranqeantityofpoplarbudsvas extractedina Instead «tractor using dier ether as a solvent. lfter five hours thebudssererenoved, groundaadthenertractedfcrsnadditionslfive burs. fhesainpcrtiencftheethersasreasvedbydietillaticn, A; A“ w’Theauthorwuldliketcthenkflrs. Shirleyoeissfcrecnducting thebieassayeonsuplesfronthisproject. W luvin‘ a dark, greenish-brash, viscous residue. The extract had an activity of 12.5 a’fil. against 5. tuberculosis. Portions of this extract one used in the follosing purification procedures. tion Three hunched grams of the crude «tract was dissolved in 300 Iillilitors of diethyl ether. The ether solution was then extracted sith five percent potassium hydroxide solution. in eumlsion formed and see broken, only sith difficulty, by manipulation. It sas found that by the use of additional ether, three fractions could be obtained: a five percent potassium rude-oxide extract, an other solution, and a gelatinous precipitate uhich us insoluble in both other and base. Tin additional quantities of crude extract (200 gram and 1‘27 Grams) sore subjected to .the initial alkaline extraction. The three partially extracted ether solutions were combined and extracted further until the potassium hydroxide solution exhibited only a faint yellos tinge. A total vole-o of six liters of potassium Inch-oxide solution was necessary for the extraction. The base-extracted other solution us washed uith distilled ester Intilthesashingsvereneutraltolihns. c , tic The neutral other solution from the alkaline extraction us then ushed with five percent wdrochloric acid solution until the ash sac colorless (volune of acid solution required use approaiaately tsc liters). The ether solution use fished eith distilled avatar to remove ludrochlorio acid and then dried over calcium chloride. The ether solution of the neutral oil we then concentrated by eimle distillation. The 183 true of neutral oil obtained had an activity of 12.5 17-1. against a. W. W92 Theneutraloilhadapalegreencolor. Thscolorresainedinths pot than the oil was subjected to a simple distillation at reduced pressure (1 am). The simple distillate had an activity of 12.5 7A1. against 1;. tuberculosis. A mm of the biological activities for the various fractions in these preliminary procedures are given in Table I. TABLE I ' umooIcn. ACTIVITIES or micnous FROM FEW! PROCEDURES _‘4 A _A.____ A ##A W W w— W wvf Test 7/11. to Sample Concentration Inhibit Growth of in Percent (If/or) $5. tuberculogig cm. Wt Do). 12.5 '0‘“ oil 0.1 12.5 angle distillate 0.1 12.5 A A‘— _‘ _a__._ _____ .k‘—_ ‘4...— ._-_ an adiabatic Jacketed colm, 1.2 on. a 57 cm, packed sith glass helicessasused for avacuus fractional distillaticnof theth oil simple distillate (prusure, 1 an. t 0.5 .11.). The aajcr portion cftheaotivenaterialsns foundtobepresentinthohighorboiling fractions. The data for a typical fractionation are given in Table II. ‘ TAKES II. DATA FOR FRACTIOHATIOH 0F NEUTRAL OIL Boiling Test 7/2211. to Fraction Range Concentration Inhibit Growth of in C in Percent («f/fir) fl. tuberculosis 1 50-71 0.1 Inactive 2 71-87 0.1 Inactive 3 87-92 0.1 ' Inactive ’4 92-99 0.1 12.5 5 99-105 0.1 . 6.3 6 105-1114 0.1 Inactive 7 Ills-116 0.1 12.5 8 Pet. 0.1 12.5 ? h 4 H L l l ____,_., fivf W ._.,'__ Chromtomhic rurification Since the antibacterial activity appeared to be concentrated in the high boiling fractions of the neutral oil, efforts were turned to the purification of these samples. The isolation methods and the infra- red spectra indicated that the high boiling fractions were terpemid in nature. Son and co-eorkers have done a large amount of terpene ieoo latioa and identification and have found chromatogrsptv on alunina to be very helpful in their nrk. For these reasons chronatOgraphy seemed a‘lcgical method of isolating the active principle in poplar buds. herons preliminary chrcnatcgrms were prepared but results were unsuccessml owing to technical difficulties, spreading or blurring or even less of activity. However, this lork indicated, as is also borne out in Table II, that the low boiling fractions (all fractions boiling bales 90°C at 1 ma. pressure) are mum that medium boiling mum (fractions boiling in the range of 90°C to 110% at 1 m. pressure) contain an antibacterial substance; that high boiling fraco ticns (boiling above no°c at l n. pressure) contain an antibacterial substance different from that one in the aedinn boiling fractions. an mm of a aedinn boiling fnction (boiling range of wife to 11h°0 at 2 pp. pressure; a: - 1.1930; activity - 31.3 27:21.) no taknnpinaainimancnntcfpetroluaetherandappliedtoazoo can Alcoa alcaina column (for a description of column preparation see Appendix I and for the exact nature of the adsorbent see Appendix III). m eclnan nu developed an petroleum ether. When the «mm solvent began absorbing in the ultraviolet region, as detemined with aback-enudelnflspectrophctcneter, developmentnestcppedandthe sole-neutron“. fhecelssnus ecu-ainedcnderannltraviclet light and divided into four section based on cones of differing fluorescence. The top section, one-eighth of the cclm, us comprised cfaredush-parpleecneandayelloescne. theseccndeeotion,” half of the column, exhibited a purple fluorescence. the third section, MW of the sole-n, us a greenish-yellow fluorescent acne. thefcnrthseetiennsccapeeedcfthereaaindercfthecolmanddid at accrues. The sections sore then extracted with etinrl alcohol. 10 a bastion Iith an activity of 7.8 27-1. sss obtained from the center cftheeclnaaasisshcuin‘rable III. rm III CHROHAWHIC DATA FOR MEDIUM BOILING FRACTION ._-.-m-——---4WW- “p---“q..—. - 'w_ _. - __ 7/m1.to Dayle Bangle Origin Boyle Weight Inhibit Growth of in Grains g. @umog 9-127 Petroleua ether 2 .019 Inactive ”hinge 3-128 Ethyl alcohol elnate 0.692 62.5 of fraction 1 3-129 Ethyl alcohol elnate of fraction 2 3.280 7.8 13-130 Ethyl alcohol eluate of fraction 3 0.392 62.5 13-131 Ethyl alcohol elnate of fraction h 0.223 Inactive ##4 __ ._._._.L W _.._.__.__- #__ A-i _‘-— Am A# A hapleD-l29 frcathis colmsasappliedcnaloo panalnaina (Alcoa) eclnm, in a petroleum ether solution. The cclnan see cashed aith petrolenethernntilthenshingsbegantoabesrtinthenltraviclet region, atshichtinethenehingnsstcpped. Thecelnanus extruded and divided into three sections. n. top em-eighth of the column was eeeticnene, theneatone-halfcfthe column-s seeticntnandthe loser three-eighth of the calm formed section three. The sections were then extracted with ethyl alcohol. The second section produced the largest fraction but its activity sas 12.5 Th1. am no lower than the activity of the parent eagle, D-129. The data for this cclnnn are given in Table IV. mm IV CHiOMTOCH-IAPHIO DATA FOR 13-129 “AA.— u. w— w ,— 9’ fin. to lasple Beale Origin Sample Height Inhibit Growth of in Grams g. mbercflgsg 0-133 Petroleum other sashings 0.006 Inactive 11-1311 Ethyl alcohol elnete of fraction 1 0.095 Inactive 12-135 Etlvl alcohol ehhte of fraction 2 2 .6111 12 .5 3-136 W1 alcohol elnate of fraction 3 0.025 Inactive _ai ___ __ AMhA __‘ A A... W W w —.—— '— v—y WW ‘1..— squmnsmatma com-u subjectedtoasimle distillaticntc obtain a tin:- vieccne oil, 13-138, Ihidh had an activity of 12.5 EV/nl. the ulecnlar weight of 13-138 was found to be 228 as detemdncd by the Rest nethcd. 1 0.0315 gran eanple of 13-138 as dissolved in 0.3166 pm of canphcr to give a melting point depression of 15.o°. l‘he acm- freesing point depression constant for csnphor in this concentration range Ins taken as 38. The specific rotation .8 found to be 01.1.85 (in chloroform). The refractive index as l.h910. The carbon-Woe!!! analysis (by the Geller Laboratories, Eackensack, Hes Jersey) was found to be 80.76% carban 11.1.55! hydrogen. 1 mar: of the physical mama of 9-138 are listed in ratio v. The ultraviolet spectrum IAELE V PHIBICAL warm 0! 13-138 W [a] 3‘ (in own.) . 1.1.35 .Holecular scight (by'Rast method) 228 se in 101‘910 Expirical formula 0,511.60,» w— w—v __ __._._.— ‘7 is shouinflgurelandthe infrared spactrunis shominFigure 2. 0n standing for a number of seeks, the biological activity of 13-138 decreased. The infrared spectrun and the decclcrisation of a potassium pernanganate solution indicated that the sanple eas an unsaturated ecnosnd. The empirical for-11a (Ouflnohfi, as calculated from analytical data, approaches that of a nonccyolic sesquiterpcne alcohol (cuHuO). It scanned possible that the excess uygen content of the isolated material ens due to peroxide formation, a sell knosn reaction in this type of cospcund. .ipercxidetestan—DB (fourncnthsafterthsinitdalisolation) astoundtobepositive. inellancuntcfetlvlalcchclnslayered On top of a dilute means solution of ferric chloride and potassiun ferricyanldeinaenalltesttnbe. Onedropcfthesaepletcbetestcd ”placedcntheinsideofthetesttubejustabcvethealcchollsycr. By tilting and rotating the tube the 88.11910 was nixed with the alcohol. A pen: to blue color at the interface of the ferric chlorideopctassinn ferricyanide solution and the alcohol layer as indicative of a peroxide. Absorbance 1.8 1.6 [\3 O l l, J l ‘ J l I l 2&0 260 Figure l. i ”80 300 320 Wavelength in RP Ultraviolet Spectrum of D-138. 4#;;::= 1 12 L 10 100 L 1 l O O O O (I) \O .3 (\J uoxsstusueal guessed Nicrons ure Liquid). (1+ 1h. Jon or biologiool activity «and to be muted uth on mowed mutations“)... Thismbomouthythotollmg Wt. mar ttondug in tho refrigerator for at lent four months, mon-uammagmmhammnufcumommu» hr. mt. The unplo no than not out for bin-lay 1nd carbon- Won Inflow-in. rho lupin 1:0an 78.28; oarbon and 11.011 lath-om. nummrmu, thoozygonoontontminormodmdthoanu- bum activity In dooromd. Boom-o of thm result- immatuon Iork on this ample m (ii-continued. rmn WWOFWMOIIGEWOFD-UB Fraction Empirical Fox-mm 3’ /n1. to Inhibit Growth of g. 1211»;eron ‘— _4_.__ _..u_ ‘_..._.__.___‘ AA.— fiw—W. w w ‘—.— w—ww w 13-138 (mm) cannon, 12.5 13-138 (after four months) 0153:4101.» 37 ._._ ._.__ A _._._ __... Author Indium boiling traction (13-123) had tho following propor- tion boiling rmgo or 99°c~1o§°c 0.15 1 m. pros-urn ootivity of 6.3 7/!1. AWofthilmmlppnedonIIOOynm (noon) oolm in a petroleum other «lotion. Tho oolm no dndopod no: pun-91.n- other until the «mm. ”mm; hogan to uboorb strongly in $110 ultraviolet region. Tho development In flopped at thil point and tho oolm m utmded. Tho column .8 divided into four muons. 16 no (hot oootion no tho top one—eighth of tho colmm. Tho second notion no tho nut one-eighth of the column. Tho third oootion was tho following throo-oightho of the column and the fourth section no wood of tho rmindor of the column. The individual sections were mud in o Soxhlot extractor using othyl alcohol as the solvent. A fraction Iith an activity of 3.9 37ml. no obtained from tho cantor of tho column as 1o shown in Tablo VII. rm VII CHROMATOGRDEIO DATA NR D-123 _._ # A A #‘A V m— w— .___ A... _ .A A_‘ A... ._._‘_ 1 u w w W WWW—"- w "w . rm. to Mylo Bowlo Origin Bonito Height Inhibit Grotth of in arm 51. tuberoulooio 13-139 Potroloun othor mhinga 0.172 62 .5 13-1110 Alcohol olnato of fraction 1 0.519 15.6 D-lhl Alcohol olnoto of fraction 2 0.353 31.2 D-th Alcohol chute of trootion 3 1.7110 3.9 13-th Alcohol oluoto of fraction 1. 1.021 . 1.8 A 1 l I H A w W __ —W Wm." Fraction- D-lh2 and D—llfl from this column mo combined and o oooond ohmnatogron no prepared. Homer, tho octivity no loot and ark no not continued on this bastion. Tho infrared spectrum of 13-1112 to given in Figure 3 md tho “violet opoctrun in Figaro h. lY .Avfidvflg mppwv NJHIQ mo Czppommm UmhmnwcH .m mnSme mcopofld S S m o a _ _ q _ 1 h _ q _ q _ r _ r _ e _ p _ _ ON on om OOH Jad uozsstmsueal queo Absorbance 1.8 1.6 1.} 1.2 1.0 0.8 0.2 I l I T l 1' l T 18 I fi[ 11 l l 1 ll t, I i i’ i. “wit J 220 2&0 260 280 300 320 wavelength in Hp Figure h. Ultraviolet Spectrum of D-1h2. 19 An 11.6 groin couple of o high boiling fraction (boiling range of o o oo 1.11 0 to 111: O at l m. proeouro, ”D - 1.139145, infrared spectrum given in Figure 5) was applied on e 350 gram almnimi (Alcoa) column in patrolman other. The column me washed with petroleum ether and then an eluted with ethyl alcohol. No fractionation was obtained as is inflected by tho refractive indiceo in Table VIII . rm VIII 0830me DATA FOR HIGH MILING FRACTION m1. Origin ' ample Weight use in Gran 8 1/2 litoro of petroleum other ”hinge 0.1116 .. 210 ml. of ethyl oloohol-Ipetroleum other overlap 0.015 1.119111 20 n1. ethyl alcohol 14.058 1.119115 30 ml. ethyl alcohol 3.721 1.119111 50 n1. ethyl alcohol 1.353 1.119115 100 ll. othyl alcohol 0.687 1.15915 ___ ___+. A; _. ‘_....i ‘— M A‘ __.-.‘ _._. ‘v w v. V—w‘vfi—fi—v V_' *— —-~— —-——— The loot rom- fractions from thio column were combined and e petroleum Other oolution of these one applied on o 300 pen alumna column (M, noutrol, gone II). The oluant we changed from petroleum ”he: to diotlwl other by grodiont olution. Three principle froctione Viroobtoinodooio ehovninroblo IX. .meflsefln mhsmv nonpomum muflaflom :Mflm co Esnpomgm emuehecH .m museum muonoflm ma OH O O A fi — d 4 — d n — l 3 J J — — _ — p F r — p Om 0: ow Ow OOH uotsstmsueil iueoneg Ill’lr' rib ill, I.- lul’ [Ii llIll 1|} f ill [it '1" 4‘“ it] 11“ ll . 1 1| gs. .. 000.0 gang .d. 0% 00.8 3 .85. .300 £93.99? .d. can :73 0.3a .85. 30.0 angina. A... 000 firm“ as 85. $38.: .. 03.0 Hoafieoefifi. . do on 38.3 «a .35.. .85. md $0.3 amino 53.50985. .4. 0% 38.3 3-2 .5 35. ms..." 30....— mmoé 3398.85. 23 03 2.8.8 0" £35. .33 33.." R04 configures». .d. 03 3 £55. 85. m3: 0.8.0 £30018? .1. 2. S 55 ~23. REA 84.0 gadget As on," on 3. ma mgfiflufi .HoHBb «mg; 30.0 53839.85». .1 com .313 $3.." mmmd .550 53933 :0. on." S 05 0 $34 03.0 .85. 533.com .0. 0m 0 .1934 330 .85. 53802“ .d. 00 p Wad $34 08.0 85. 533$ .3 cm 88.5 0 mg: mad Bet Beanbag .1 2. m a 08.0 .85. Eng .1 8 a..." owned—5.8a .z 3.8 5 «o 5:80 333 00 an»; .33... 53.8 .3 838.... nggmgdgonugg “a 22 FmtionoSond6nrooobinodto¢ivooomploD-210,trootiono7to16 todvoouploD-fll,ondtrootionol7to19togi.vooonploD-212. All throooqloouorothonoubjootodtoooimlodiotillotiontoobtointhe Iotu'iolom'rouoodtorthofonouing lurk. ‘rho murochlorido dorivotivooctW, momaZvoroproporodinthorolloungnonner, oooordinztothonothodot‘éorn (13). 10.2 “moumooiim diooolvod in ton ill. of obooluto diotlvl other. The oolution no cooled to looo than 0’s in . colt iooboth end thou ooturotod with nhydrouo Wm moi-ice. Tho oolution turnod from light yellow to dork rode bro-1. Tho oolution no kept ot ice both mature for 36 houro, thonthoothorondhydmgonehloridourormodgm(nkingnro thot tho tonporoturo never rooo above 0%). no relating, cryetelo core “the nininnonountotoolduoflvlolooholondthenmmtol- liood for notle alcohol. the yield: ooro between 50 and 75 percent of Wm. The melting pointo or the We“ at ouploo 13-210, 211, m ooro 78°C (unoorrocted). Tho opooirio rototion, rotroctivo in- 4.. end nativity or thooo traction m oloo dotoroinod. Tho corbon- We“ molly-co were no follow D410, 80.822 carbon, 11.833 hydropn} 11-2111, 00.57: oorbon, 11.61% moi-om m, 80.50: oorbon, 11.711 bureau. Innthioinromtionitoppoorodthotollthroooolploo, D-alo,mondm,ooroprimnythoouoconpound. mointrorod Wormwouqnoo, giminl‘igurooé, 7end8,oloooupport thioomlnoion. Amorthomoioolproportioootthooo trootiono to chain mu. 1. A 4), NH .Amflsz. onsmv OHNIO mo Ednpommm oopwnmoH OH m .w mesmflm mGOMOHE —\O .u-u—-—.—._——_ ON on om Ow OO uotsstmsuenl iueoaog 4. emoflq mnomv Hams: mo Ednpommm copmcmcH .\ opzmwm c 2"1 mcopofl: ca w e e _ _ e q _ _ _ q 1 0m 1 Q: i 8 p L _ r _ _ _ ooa wsueu; queoaed L 88" "K‘ U0 ‘J L m 1 _-__ T I r 1 __ —d _ _. :1 i—- —1 C) 1—' — r—l _‘ CI.) p 3: <:::'—7 __ p— F— —q\.(\ F — 1-— _..:;t f _ :3 If 1 1 1 1 O O (J O Q g 0;) \Ca ,3 N uorsstmsueal hiorons i). 'r \y 26 rm 1 ’HIBICAL PROPERTIES AND ACTIVITIES OF D-210, 211 , 212 AMA __._. _‘ #4.- AAA-#AA m 1‘n [“1 n Hydro- Inhibit Growth of Form]... chlorida 311.. W n-m 1.1969 950.9 78°C 6.3 Ouflu°xm ”.211 1.11952 .5302 7800 603 0153.003.” 9.212 1.1.960 osm 73% 6.3 01533001.” ——‘——v— _.—,v aqua 13-219 m roohronto graphed undar oonditionl comparable to the m column. The unplo an analogous to 11-212 in chrmtographic m. Omnumlndormthil mt the mun «hummer. withumploD-mbdnganmmotthowpliodulplgmmt lupin 13-212 in a rohtivoly pure couponnd. 81300 the «perinatal realt- 1ndiontod the ”the principlu 1n Wuwdtruuonotpopmbudoflmtmcmum,n warmthmmimfiothutorpmeainmnpouua Inch mum ”unity. Accordingly, :11 radily «mama tarpenaid Wmmtoruuutytgdmtamorhcm, Wm'mmwmw man-«WW. mmwmm mrmrdodin‘hblon. IABLEII 27 mm OF SEVERAL WEEKS AGAINST BACTERIA *— A“ __L_ wfi. ‘— W _ ww— WW w “AA fez-pane Hicrococcue Salmonella lanthomonaa bum-bacterium pycgcncc, typhimriun phaeeeli hibercnlccil aureua a I f I“: 7 "1' I 373' 2731: Oineol Inactive Inactive Inactive Inactive Gitrenellal .310 Inactive 310 62 Citrcnellol 310 620 160 62 Geranicl 310 620 310 62 Linelool Inactive 620 620 Inactive d-Lininem Inactive Inactive Inactive Inactive 61-111mm). 310 620 620 62 ldnthol 620 Inactive 620 62 Pine!» 620 Inactive Inactive Inactive Pulegcne Inactive Inactive 620 Inactive Iqualene Inactive ‘ Inactive Inactive Inactive x-rerpinecl Inactive Inactive Inactive 125 Minn Inactive Inactive Inactive Inactive tarmac]. Hot tented Not tested Not tested 31 _.4._.__4A A _.,___....v 28 DISCU33ION “mucnofthedataini‘ablelccnldcanaeenetennderir aw Nation or fractionation had been effected by the alkaline at! acidic extraction. Homer, Corie and Oanal(12) ieelated and idutitied acne cinnanie acid derivativea from an alkaline extract of poplar bud oil from 1. Wm. A umber ct cinmunate derivativea, vereteetedbylucae (nnpubliaheduork) and macro foundte have etc-ens antibacterial activity. In all probability, thee-atone, active eimaaetee were eeparated from the terpeneid ccnpeunda on which when: he! been placed in thin work. Fractional dietination of the nutral 01]. indicated the We ettvedittemtantibecterialnbetancee. Wdththenterial mun fractional dietillatienvitheboihmrange o: 10h°c to 1:130 atln.peemredidnetyield reeulta niche-recenclueiveaatcthe exact nature of the active principle. The infrared epectmn (1h). ultraviolet epectm, optical rotation, mm: o: refracticn (15) and carbon-Ivdmzenanalyaiedidindicate thatthenatec'ialnaaeeequio Wenonah Simetheaatarialueaaectainedtcbetmmcin nature, it use logical to aeme that oxygen could react with the anaetuated carbon atom of the molecule to tern pea-exidea. Ihie point ”tormentbyapeaitiveperexideteat. Achangectthiatypein the aelecelar etmctnre er ecnpeeitien night he meted to alter the antibacterialprcpertiea etanaterial. Inthepreeent eaaeadeereaee in activity did occur, ae ie chem in Table VI. Becanee ct theee 29 dittienltiee, ark on the active compound in the medium boiling frac- tion we diemtinned. 1 puma: my; boning traction (mm) is believed to be an incur oi’ biaabolol (I). The preparation of a lvdrochloride (II) thich ‘8 identical in uniting point lith the Wehloride obtained by Sam (13) from an authentic mp1. o: bieabclol ie good evidence for on: eenelaaion. OH - 01 Cl ‘. I II The refractive index, apecific rotation, and carbon-morocco analysis at 11-21: compare favorably aith thoee vamee tor biaabclol, aa ie m in table XII. rm III PHIBIOAL PROPERIIE 01“ 13-212 AND 3131mm. --~—-~.-—-—— m ----Oea-O~n _-....... --"-a ”two..- — .- W *~~.-v-mt- v-n-ma-P-nm w—- '“W mw~ ~-....-A..-l—I .-.--- 9-212 1.1959 951.8 80.50 11.11 mm 1.11919 (Euro) (12) .51.? 80.68 11.58 1.11936 (Ruaicke) (16) A A#~A4.— w _v v —v' fiv— v w 30 The infrared epectrun of 13-212 (Figure 9) aleo comparee favorably with the infrared Spectrum o: biaabelcl (Figure 10) (13). In view of these finding it we: concluded that hisahclol ie one or the major active principlee in the neutral fraction of poplar bud oil from 2. “0810811803. It ie alec possible however, that a small amount of impurity could account for the biological activity or this fraction. Final and anewivocal proof would require more). etepe. First, isolation of biaahelcl from other plant aourcee and then teating the commie for activity againat g. tubuculogi‘g . One ecurce c: hieahclcl, according to levee (17), ie Cabreuva 011.. Born and cc-eorkere (18) ieclated l ~hieabclcl,£ron oil of chamrdle. A ample o: the latter oil no m for WW ”unity and round to be negative. l'hie brings to light the possibility that the “(1" form of bieaholcl in active mile the '1 " form in inactive. However, the actual isolation and teating or the '1' tornianeceeearyto eatahliahthiepoint. 1mm etepinproc: of the antihacta'ial activiwei‘biaabolcl wouldbethe eyntheeieoi'the compound. Ihie eyntheeiehaebeen deecribed by 3mm and Liguori (16). However, this product would be adxtureottcuriacmere inanunknounprcporticn since therearetwo aqmetric mtere in the molecule. Therefore, the ayntheeia alone wuld not cmtitute lull proof for the antibacterial activity of any m epecifio iecuer of bieabclcl. ' The teeth; of avarietyct tempemid compound- tcr antibacterial activity daaonetrated that although any or theee poeeeeeed activity, a emidu'ahle number-were inactive. The tel-pone wdrocarbcne teeted .«2 Amflzvflq onsgv .mHmID mo Sappooum @mnmnmmH .m onddflb mconoflg ma 3 m, h a q I _ _ _ . _ _ _ _ _ l.0m : 1 1 om L 8 F _ r _ _ ? p a F _ _ OOH ed uo:ss:msueal QUGOJ 12 13 ll 10 Mlcrons \ bolol. (-3 U) ' H1 {13. 'um of H +) C; Q4 (0 d C) Infrar Figure 10. I <3 :3 <3 <3 co \0 43 l uolsstusueai luaoaed 33 tare inactive whereas the terpenoida containing oxygen, in general, poeeeeeed activity againat one or more of the organism used in the teat. War notthie ccnclueionia morallyvalidrequiree further lurk. None of the complee tested, however, approached the activity of the eample of biaebclnl iachted in the preeent uork . PART II INVESTIGATION OF ANTIBACTERIAL PROPERTIEB OF THE FLOWER OF HIPERICUM PROLIFICUM 3h MOTION to far back ae the tine of Galen and Dioecoridee curative powere mattrihatedtotheplantmricua. Duringthendddleageethie plantnanentionedtdneandagain,aoetlyuaprotectiveagaimt ntehcraft,butalaoby0eraanand1rabicphyeicianeaearenedyagainet h-orrhagee,kidneyetcneeandinteetinalwhme,aeuellaaatreatuat efuande. lngliahherbalieteherealeeueedelivecilertracteof mumdwwumo). lent to nothing m known about the nature of the curative agente eprlautwhenEerw(l9),in1911,publiehedhieacocuntabcuta redpigaentuhichheealledhypericin. Ihebielcgiealintereetinthie Wmarceeedinpartioularbythefeetthatiteaneitiaee light-mmtoirradiation (20). Itappearedthatthilbic- legieally active eubetance light alec be the agent which we reaponaible forthenedicinalpropertiee. the atructure ofthiecomcunduenct Montana when Brochanfig. (21) characterised hypericinaa hemalvdrmdinothylnaphthcdianthrone. ~ Beaver, it became cbvieue that other ecnetituente of thie plant alec had biological propertiee, although clear dietincticn that the varioue effecte were due to different ccmtituente waa not made at firet. ‘l'hefiret public reportabeut the antibacterial activitycf eradeeztraetacfthieplanteanefronJeneenandHiller(22),ilc repertedntivityagdnflWmindilutiommtc 112000. Iheee nrkera dealt with rather crude preparation which, in 35 addition to an antibacterial principle, also contained other mbetancee , a for instance mericin. While all of these literature sources dealt with the species 11. perforatun (a weed, commonlyr called St. John'e firt), Lucas and cc-mrkers investigated several species of the genus m for antibacterial proportion and first reported in 19h? (23) considerable activity of leaf extracts of g. cgl'ginum against 3:1,. m. 1353. Later, the same group reported strong antibacterial properties of extracts of leaves and particularly of flavors of g. perforattm and gmlificun(10)andcf flowereofg. nceeriammandgefiw (no). It the some time, Regen-trad (25) found an unidentified anti- bacterial agent in capsulee of g. mforatmn. In view of the foregoing information, a project he initiated to isolate and identify the antibacterial principle“) in g. prolificun. 36 human AND RESULTS 31mg; file principal test organim in the work with Mariam: prolific“; he fimccug may; var. m. However, a yam-negative organ— iu, 28.11ng phaseoli, was also used in the testing. The activity against g. @6011 was generally higher than that against 11. mm but in order to simplify procedures only the activities against the Maths 1!. agreea es were used in the follovdng experimental work, results and discussion. The bioassay was accomlished by the aerial dilution method (Appendix II). A discussion of the methods of express. in; and calculating the activities may be found in Appendix II. Winn-«2.1. neblcsmscfg.m;i_f_i_cu_gmchwereusedinthisnorkwere obtained by Dr. E. H. Lucas from cultivated shrubs you in the horticultural Gardens of hichigan State Mversity. The shrub produces anabundanceefflewersdu'ingthenonthefhly. Theharvestedflonere were stored at alo°c until needed. Mn oLthgjrude Egtract A study uas first undertaken to investigate the efficiency of vari- ous extraction methods and solvents. helium unpublished work by Lucas indicated that the three nest suitable solvents were ether, petroleum ether and ethyl acetate. To put these preliminary results on a quantitative basis the follouing procedures were followed. 37 A 15-pin quantity of flavors was nacerated in a Haring Blender, using 150 ll. of the appropriate solvent. The extract was then filtered and a portion as sent for bioassay. A Soxhlet apparatus was used for a seeeni method of extraction. The lS-gram flower earple was placed in the extractor along with 150 ml. of the solvent and refluxed for two and one-half hours. The Waring Blender-ethyl acetate combination gave the highest amunt of activity as well as the lowest amount of total solids and therefore the extract or the highest specific activity. These data are given in Table XIII, TABLE XIII RESULTS OF VARIOUS EXTRACTION PROCEDURES WITH EXPERICUM FLOWS A “ ##__“w WA“ “ w—w .'_. w 1, ,7 #— T ——.——.—— ’— fi— W ____.. _.___‘ A A... #H#——-‘A—n— _._.. w— w ww— ._T__._ 'Zrl/xdl. to Mention Extraction Total Solids Method Solvent in Gran Inhibit Growth of Es 2221561198 lezhlet ethyl acetate 0.975 S Seahlet ether 0.870 2 .5 Sublet petroleum ethm' 0.750 8 Waring Blunder etth acetate 0.1.35 0.6 Haring Blender ether 0.750 h Vanna Blender petroleum: other .A‘ A. A___._..__‘_‘ V __...— ww— w __A W— o.535 6 ‘w V-v.’ Forlargescalepreparatimtheuseotawaringnlendorusin- practicable: thustheprecedureactuallyuedintheupa-imtstebe described involved mention of the {losers in a food chopper 38 followed by extraction of the mcerate with etmrl acetate. The follow- ing is an ample of a typical preparation: in BhO-grsn quantity of flowers was merated, covered with two liters of ethyl acetate and allowed to stand for one hour with frequent, thorough agitation. The ethyl acetate was drained frcm the macerate and the extraction was repeated two additional times, using one liter of ethyl acetate for each extraction. The individual solutions were combined to give the crude extract. The activity in this particular case was 2.0 7/ml.,‘ however, the value varied with different preparations, between 2 and 10 find. Some variation in the degree of activity of the crude eortract ins noticed, depending upon the year of harvest, the stage of flowering at the time of harvest and, of course, the method of preparation. much ofWBtock Solution The crude extract was taken to dryness in a flash evaporator, leav- ing a residue of approximately 100 grams. Attempts to purify this residue by use of different solvents were unsuccessful. For maple, the residue from a cmde «tract was dissolred in a quantity of ethyl acetate equal to 0.05 of the volume of the original extraction solvent. This solution see decanted men the residue, diluted nth diethyl other until precipitation ceased, filtered, taken to dryness in a flash quorater, redissolved in other and then filtered again to give the final stock solution. However, it was found that activity as lost at eschstepiutheproceduresndthatansanplesmenaposedtoair 39 no subject to an intense browning reaction. is a result, the final method of preparing the stock solution consisted of simply adding 100 ll. of other to the residue from the crude contract and letting the solution stand at 8°C overnight. The solution was filtered and the residue lashed 111th 100 ml. or other. The washings were added to the filtrate. Tho biologically inactive residue as a green powder which we a positive Holisch test, indicating the presence of a carbohydrate. the filtrate prepared in this manner was designated as the stock solu- tion and had an activity or 1.25 3 /m1. However, the activity or different stock solutions varied from 1.25 37m. to 10 7/m1. fidic and AlkalineLExtracticn gfjhe Stock, Solution 1 20-1111. aliquant of a stock solution was extracted consecutively Iith three 50 :1. portions of 0.1 normal acetic acid and three SO-ml. portion of 0.1 normal amcnium hydroxide. The acidic and alkaline Wants were taken to dryness and dissolved in etln'l alcohol for bio- assay purposes. The activity or the original stock solution was not shanpd by acidic and alkaline extraction. The acidic attract was in- active and the alkaline extract did not exhibit appreciable anti- bacterial activity (Table XIV). In another experiment using 0.1 normal hydrochloric acid and potassium hydroxide, some activity appeared in the alkaline extract. no acidic extract as inactive, and the residual other solution shoved a loss of activity (Table IV). It was concluded that the active principle could not be satisfactorily isolated from the stock solution by acidic or alkaline extraction under the oorditione of the experiment. ho TARP} XIV 310133;! mm 0N ACIDIO AND ALKALINE EXTRACTION or STOCK SOLUTION USING 03,0008 um 311.011 ‘3‘ “‘ A ““ ' “é *’ Samplo I /ml. to Inhibit Growth of 210 22283119. Stock oolntion 1.25 Asidio txtract Inactive 11min. utruct 320 Stock Iolntion.a£tar acidic and alkaline cxtraction 1.25 TAHLE XV BIOIBSAI’DAIA 0N ACIDIC AND ALKALINE EXTRACTION OF STOCK BOLUTION USING 8C1 AND K03 A AAA A __.___ ‘- _.__.__ _——‘ A A ___.__. _____ A‘ aalplo 775fl» to Inhibit Growth of 2!. w stock solution ' 2.5 lantrnlisod alknlino extrgct 10 Acid «tract Imotivo Stock oolntian Artur acidic ind alt-lint attraction, 7 A “#— _A. __—-.‘—____._.n—— .._—. __. W— v—-—~ v— _— w 7' VW fleetiongg Adsorbent Since further solvent extraction did not aid in purifying the stock solution, it was thought that adsorption cinematography could be helpful. Using several different adsorbents (Appendix III) columns were prepared in the manner described in Appendix I. The results of these trials were such that the adsorbents could be grouped in three classes. Some, such as Alcoa alumina, Woelm neutral alumina, and nagnesia, adsorbed the active principle so strongly (Table XVI) that it could not be removed, even by prolonged treatment in a Soxhlet extractor. On the other hand, another class of adsorbents composed of calcium carbonate, cellulose, silica gel and starch, had so little affinity for the entire sample that it passed through the column in the first felt milliliters of the developing solvent (Table XVII). The third class of adsorbents consisted of Celite, pH 2 alumina and pH 1.; alanine. It was possible to apply the sample on columns prepared from these adsorbents and then successfully elute the antibacterial activity tron the colunn. Ls night be enacted, all adsorbents did not [all into Just one group. Bork lith charcoal produced varied results. In one case, the saIple was applied on the column in other and then eluted sith ether. the sample had an activity of 0.01 77ml. Efforts were nude to reproduce this unis but all attempts failed. In all or the following chromato- p'ans prepared using charcoal as the adsorbent, the activity either could not be removed from the column or it see last when the sample ‘0 renewed by extraction in a Soxhlet apparatus. The conditions for 1&2 TAKE :71 ADSORBEN'I‘S WHICH RETAIN ACTIVITY FROM HIPERICUH STOCK SOLUTION w—— W ....._ ‘- ___..‘ _‘____ - ————— V w Colunn Adsorbent Method of Preparing Solvent Used to Number Chromatogrsn Remove Activity I Alcoa alumina extrusion ethyl alcohol II Alcoa alumina eluticn ether ethyl alcohol XIV We elmn neutral slution and ether almina extrusion ettwl alcohol ethyl acetate acetic acid in ethyl alcohol ammonia in ethyl alcohol XV Magnesia elution ether ethyl alcohol TABLE XVII museums man no no: arms ACTIVITY mom menace srocx common M “Lg—h A - A.‘ _.._ MA M M w __4__ AA A ._.A‘ _..‘___ w *— w I... Column Adsorbent ' Method of Preparing Solvent Used to Mar Chromatogran Remove Activity III Calcium carbonate sluticn ether VII Cellulose elution ' other xx Starch elution ether III Silica gel elation ether __ purification of the stock solution on charcoal must be quite specific and sufficient ties for further investigation of these conditions was not available. Consequently, work with charcoal was discontinued. Chromato ggphic Purification by Elution mm work indicated that purification of the stock solution by two step adsorption chromatography, using pH 2 alumina followed by pl! 1; alumina, was very effective. Both elution and extrusion chromato- graphy were used in order to see which method would give the best resolution of the stock solution. A 23.14-gram sample of a stock solu- tion (activity 1- lO i/ml.) was applied to a 615 gram alumina (Woelmn, pl! 2) column. The initial eluant was other and the fraction volume was approximately 300 ml. The ether removed a yellow-green zone containing about 1.5 grams of sarple. The most active fraction in this acne inhibited bacterial growth at a concentration of 31 7’ /ml. After allow»- ing 1500 Il. of ether to pass through the column, the solvent was changed to two percent ethyl alcohol in other and eluticn of a dark-green sone followed. The total weight of solids in this zone was about 9 guns. The peak fraction had an activity of 1.7 z’fnl. One liter of tn percent ethyl alcohol in other was followed by two liters of ethyl alcohol, eluting a green fraction (three grams). The activity of this last fraction sues over 202’ /ml. The data for this daromatcgram are given in Table XVIII Fractions 9 and 10 from this chromatogram were combined and applied to a 31:5 yam alumina (pH )4) column in ether. The column was developed rm XVIII DHLFOREUTIOICmeBZALm +V~f f ‘v' .A— i __L h. _‘..._ A“ *— 7 .7 7m. to Fraction Volume Solvent Total Weight in 111. in Grams Inhibit Growth of .11. mgenes Stock solution ether 23 .h 10 l 300 ether 0.026 Inactive 2 206 ether 0.050 Inactive 3 230 ether 0.2M Inactive h 285 ether 0.765 220 5 300 ether 0.20? 31 6 280 ether 0.126 kl 7 310 ether 0.100 5 8 310 2‘ etlvl 0.108 15.5 alcohol in ether 9 320 2% othfl 5 .075 3 alcohol in ether 10 11.0 21 ethyl 2 .027 1.? alcohol in ether 11 320 2% ethyl 1.102 3 .6 alcohol in ether 12 11:0 etlvl alcohol 0.3h0 SJ: 13 300 ethyl alcohol 2.063 22 lb 300 etlwl alcohol 0.571: 31 15 1500 ethyl alcohol 0.5115 210 its and eluted with ether which removed one com, the most active fraction having an activity of 7.8 771111. The acne containing the major portion of the active principle we eluted with a one percent eolution of ethyl alcohol in other. The mat active fraction was umber 13 which had an activity of 0.25 1/nl.‘ The data for this column are given in Table III. rm: :1: Din ran manor cmomrocniu men pH 1. mm n.- . Y/nl. to Motion Volume Solvent Total Weight Inhibit Growth of 111 me in cm as m l 270 ether 0.081; Inactive 2 320 other 0.110 31 3 320 ether 0.076 31 h 320 ether 0.068 15 S 290 ether ‘ 0.051: 15 6 320 ether 0.063 15 1 310 ether 0.015 15 8 170 ether 0.0.35 7.6 9 320 ethyl alcohol 0.071 7.8 other 10 320 is etl'wl alcohol 0.053 3.9 in other 11 320 {S etIvl alechcl 0.0” 3.9 in other 12 300 15 etlvl alcohol 0.th 3.9 in ether in other 11. 300 ix ethyl alcohol 1.21.8 0.50 in ether 15 370 11 ettvl alcohol 1.610 1.0 in ether 16 500 11 ethyl alcohol 0.370 3.9 inether *_._‘ _.._- 146 An attempt me made to reohronatograph the combined emle of fractions 13, 11;, and 15 on pH 14 alumina. In the process, however, the activity on lost. rho ultraviolet spectrum of fraction 13 (H-282) is than in Figure 11 and the infrared spectrum in Figure 12. Chronato Egghic Purification by Extrusion The stock solution separated into a number of sharply defined eones when applied on a p! 2 alumina column and developed with ether. For this reason it no believed that extrusion and sectioning of a colon of this type could produce a major purification of the stock solution. To this and 1.62 grams of stock solution (activity I- 1.2 7M.) as applied on a 100 gram alumina (Hoelm, pH 2) column, which us then developed with ether. is derelopnent of thecolumn progressed, msonuwidenedandncwones forncdeothatwhonZOOml. ofether. had passed through the column the aches were arranged in the following order from the top of the column down-rd: a blue-green acne, a dark-v peensone, eyellowecne, aclearareawhichblendedintoaligbt- green acne, a narrow acne of a bright red eaterial, a blue-green acne and a yellowsone. A yellow zone which passed through the colmm with the ether as found to be inactive. The column was then extruded and divided into three auct- sections. The first section, a-hha, contained the top three sense and part of the clear area. The light-peso acne, the red acne and the blue-m acne mend the aecond section (Ht-1.1.9). meyellewscneandtherenaiuderofthecolunnnadeupthethird section (3-150). Each section was then extracted with both ethyl alcohol and etlvl acetate. The extracts were combined, taken to dryness, Absorbance 1.8 1.0 0.8 0.2 Javelength 1n LP Figure 11. Ultraviolet Spectrum of H-282. r I Awflsqu mndmv .mwmum mo adipommm JmhmnmsH .NH onsmflm msopOflm S on m o n. _ _ _ a i q ‘ _ _ _ 1 8 low _ p _ _ P _ r _ p _ 03 uoisstmsucxm quaaied and redissolved in other. the activities of the extracts of those sections were as tolloas: section 1, 15.6 7/1111": section 2, 0.8 271:1... section 3, 0.72 f/nl. A smry or the data for this chromatogran is given in table xx. ‘ TABLE II DATA FOR EXTRUSION OWNER”! FROM pH 2 ALUMINA 77ml. to WW.— w— ———.~ *7 ——~ Fraction Origin Inhibit Growth at 9.!- 212m l-Mi’l 132 ml. of ether elunte Inactive we Ethyl alcohol and ethyl acetate 15.6 extract e: section 1 Edit? Ethyl alcohol and ethyl acetate 0 .80 extract of eection 2 3-150 Ethyl alcohol and , ethyl acetate 0.72 extract or eectia 3 Fraction 3-1450 no chromtogrephcd on a 2S-gran slmIina (pH 14, grade II) oelnnn. the ample as applied tron ether. The developing solvent, ether, eluted an inactive yellow acne, 11-1351. Previous 1nun-k indieatee thatapfihelundnacolunngavebeetresultsldimtheelntion uthoduued, thnstheeluantsuchengedto ethylalocholbythe padient aethcd and a second yellow anew-162) was removed from the eolm. rhi- traction hed an activity or 0.1.2 7/:1. The data tor this oolm are given in roblo m. 50 um: III DATA FOR CW 0? 8.1150 _A A‘ A...__. _.A _..._ __ _._ .._.. .-_ -- ##A A“ A“ ..._~ *w!‘ A“ 77nd. to traction Elnant Volume total Weight Inhibit Growth of Solvent in 141. in Grams 311,. m Bowl Etha- 125 0.2147 Inactive lots: Ethyl alcohol 100 0.080 0&2 A A .A ._. _ .M w w." w v— w w— Fraction B—ldi9 was also chromatographedona 25-pan alumina (pH 1:, paden)oolnnn. Theeanpleuasappliedontheoclumi‘rmanether solution. rheolutioooothodeueleouodrorthieoolm. emu-nu eentinsdastheelnantandtneoneeeereeluted,H-h53andH-h5h, bethotshichsereinaetive. melantuohongedtootmeloom bythegradientteohniqneandarellovtraetion(K-h56)usthnselnted. rheaotivity oithiotnotionueoal 77-1. the data forthis ehronatograaareginnini‘ablenn. rmnn -_ wimcmoxmmorm _-A__.. A... An. .4“ m. .wfi W ‘- —. ._._n “A #A m A AAA—‘AA W W. Wm— Iraotion Elnent Volume total weight Inhigfirorth of Solvent . in El. in Ora-s m 8-163 rum 5h 0.008 Inactive 34.51: Ether 29 0.006 Inlofl." n-hss Ether alcohol 3h 0.003 Inactive 8-356 Btlvl alcohol 23 0.033 0.31 3-15? Ethyl alcohol ho 0.010 0 .78 we mm alcohol 29 0.003 0.78 A __.‘__ _. __.‘_ _ * vw— ‘— n—iw ‘v— f 51 It us thought that the active principle in section 2 and section 3 of the poi-out pH 2 alumina oolmm the same material. For the purpon of comparison the ultraviolet spectra of 3-152 and 8456 were prepared. The qeotru or H.162 is given in Figure 13 and that of 3-156 in Figure 11.. Olalitative Intonation Concerning Bone Active Preparations gtabilitz in Heat and Air in other solution of a sasple eith an activity or 3.9 f/Il. eae allovedto etendet 37°c for eight hours. The activity ortho sample npenretestingnsfonndtobeléa’lnl. Anotherpcrticnotthesane sanpleeasalloeedtostandatroonteuperahu‘etoreighthoursuth airbabblingthrcnghit. Thebioasaeyonthiesupleeastcnndtobe 16 7/:1. The original sample stood at room temperature for eight hours ondundretutodetillhodeoootivityora.9z’/el. MW definitely bear out results vhioh eare observed throughout all puriti- cation procedures. ili 801v Grade extracts of the flowers were prepared esing nativl alcohol, ethyl alcohol, ieopo-opyl alcohol, butyl alcohol, and ethyl acetate. the extracts ears tested for antibacterial activity men they eere first preparedandthenaseoondtineISnenthelater. mes-plume stored in sealed containers and at a temperature of 8°C. It In tonnd that the activity was greatly reduced in methyl and ettvl alcohol, Absorbance 1.8 1.1: 1.0 O.h 0.2 \‘r 1. {\J Wa*elength in My Figure 13. Ultraviolet Spectrum of Absorbance H e (3') 1.6 0.6 *1 I I 41 l I 4T I ll 1 I 1 1 1 1 1 1 1 l 1 4 1 2L0 260 280 300 320 3&0 Wavelength in Hp Figure 1h. Ultraviolet Spectrum of H-hSo. 511 .113th reduced in isopropyl alcohol, and not altered in butyl alcohol and ethyl acetate. The data for these tests are recorded in Table um. ' rm XXIII mrmm. ACTIVITIES 01‘ CRUDE mm AFTER LONG STANDING Initial Activity _‘ Solvent - _* # Activity“; A After 15 33‘3” flatly}. mohol 112000 1160 M1 alcohol II 2000 1160 Iaopropyl alcohol 112000 111000 Butyl alcohol 182000 112000 Ethyl acetate 112000 112000 __.___.‘ ._._‘ H_k A“ _ L— L mu A l... fiw “—w—W—w “WW W 'creetoet diluticnwaxhibiting inhibition or g m. Although other extracts were not tested under carpal-able coalitions, routine stock solutions or the active material in ether solution main-- tained their activity for periods of at least three months. W Several sodium fusions were prepared using portions oi‘ active fractions. The test solutions obtained tron these fusions were used for elemental analysis Ihioh gave the rolloeing results1 sodium nitro- prusside test for nitrogen, negative; lead acetate test for euli'ur, neptive) silver nitrate test for halogens, negative. is a check on the Man, a [Jeldahl nitrogen determnation us also made on an active traction lith results idioating the cowlete absence of nitrogen. 55 DISCUSSION The purpose 01' this research project as initially outlined m to isolate and identify the antibacterial substancc( s) in the {lovers of m prolificun. However, automating circumstances, such as the instability of the active principle, have kept the author from attain- ing the initial goal. It has been possible though, to obtain a fraction Iith a very high activity against gigroccccus m. Considerable intornation has been gained concerning the nature and properties of the active principle which may be of help in the future isolation of the antibacterial in this plant. The preparation of a highly active traction my be mind as follows. The nacerated flours were extracted three times with etlvl acetate. The ends contract was then taken to dryness and redissolved in ether, producing the stock solution. The stock solution was then appliedonapnzaluninaoolum. itscpercent solntdnnorethyl alcohol in ether eluted an active motion iiioh us rechrcnatczraphed on pH 11 alumina (grade II). A highly active traction eith an activity or 0.3 7/111. could be eluted tron this column by neans or a one percent solution of ethyl alcohol in other. In the course of this research, some interceting qualitative inform- ation was learned about a number of. active fractions. The infrared spectre: of a highly active traction, prepared by the procedure Just described, indicated the presence or a hydroxyl group (maidmn at 3 )1). 56 an aromatic nucleus (maxinm at 6.25 p) and a carbonyl group (mazdrnum at 5.79 p). i phenol test with ferric chloride was found to be positive for some active fractions and negative for other active fractions. For one active fraction the ultraviolet spectrum exhibited a mammal at 290 um and for another active fraction the nuimn was at 270 um. All of this information can do little more than give an approximate idea as to what the nature of the active material(s) may be. However, the phenol test and the ultraviolet spectra some to indicate the presence of rare than one antibacterial material. The active material( s) apparently do not contain nitrogen, sulfur or halogens. It can also be stated that the active principle is some- shat acidic in nature as it can not be eluted from alkaline alumina but can easily be eluted from acid alumina. 57 SUMMARI l. A neutral oil was prepared from an ether extract of the buds of g. tacanahaca. A medium boiling fraction of the neutral oil was chromato graphed on alkaline alumina, yielding an unidentified sesqui- terpene alcohol which was active against 31. tuberculosis. A high boiling fraction of the neutral oil was chromatographed on neutral alumna. In this manner it as possible to isolate a sesqniterpene alcohol Idiich sas inhibitory tennis 2;. tuberculosis and as identical with ot-d—bisabolol in respect to the Ivdrochloride derivative, the infrared spectru, the specific rotation. 2. A stock solution we prepared from an ethyl acetate extract of the flowers from g. mention. A fraction sith strong activity against ~ 5. mamas and 5. phaeeoli ens obtained by chronatographing the stock solution on pH 2 and pH h alumina consecutively. The exact nature of the highly active fractions was not established but they were sheen to contain an aromatic zealous, a hydrcayl m and a carbonyl group. The absence of nitrogen, sulfur and halogens see also desenstrated. 58 BIEIOGRAPHI l. hosig, A. and Schram, Die irmeipgaggen-und Drogenschats Chinas and die Bedeu des P011 is ac hang-gm, Beihefte Der 'Pharmaaie , fid" no 1933: P0 he 2. Ebbell, 8., translator, I’m Ebers, Oxford University Press, 3. coo-ore, J., The Herb of Gener Historic g Plants, Norton and “”01... L0 , 1 3 3 Pa e h. florsy, H. m, Chain, 3., Beatley, N. 0., Jaungs, H. 1., Sanders, i. 0., Abraham, E. P. and Florey, H. 3., Antibiotics Oxford Uni- WV "0", New Iork, no 1., ”1‘9; ’0 33L Se Link Ks Pep “8.11. Be Be ‘nd Walker, Jo 0e. Jo 3101‘s Chan, 1):, 369 31933). 6. lraokner, B. 11., )1qu, Basel 11., Schaffer, P. 3. and Fontaine, r. n., J. cm. Invest., 3g, 89!. (191.9). 7e M‘t. We Be, m M as, Wt”, Po as M roam, 1. De, J. m. mute, 25., 899 (191499. 8. Matson, G. 1., Rauve, 1., Bugihara, J. M. and Burke, H. J., J. Clin. Invest" 39,, 903 (191:9). 9. Frisbey, Ardeth, Oottshall, a. r., Junings, J. c. and Lucas, E. 11., Marten-1y Bulletin of Michigan Agricultural Earpsriaant station, Michigan State College, East Lansing, Vol. 38, no. 2. 279 (1955). 1.0. “*0, m0“, Rab“, Je ne, Jflmlngl, J. 0., 00%“, Re Is and Moss, E. 11., 11:14., Vol. 35. lie. 3. 392 (1953). n. Hakao, 11., J. Phara. Soc. Japan, no. 513:1. (1921.). 12. ((10ng i. and Canal, 11., Bull. soc. chin. France, 5. Series 331982 193 . 13. ion, r., Vrany’, a. and Hal-out, v., Chm. now, go, 36).. (1952). 11‘s O'comr’ Re '1'. 3nd GOIdbhtt’ Le ‘ve. Me Chum, gill-é, 1726 (1951‘). 15. Gunther, 3., The Essential Oils D. Van Hostrend 60., Inc., In York, I. ., , o . , p. Bl. S9 16. Rusicka, 1.. and Liguori, 14., Helv. Chin. iota, 15, 3 (1932). 17. Haves, L, Helv. Chin. iota. 11, 1.08 (191.8). 18. gore, 1., Zaoral, K. and Herout, V., Collection of Czechoslov. Chen. Comm, L6, 626 (1951). 19. 6m, 0., Hoppe-Beylsr's z. physiol. Chem, 12, 371 (1911). 20. mm, L, Klin. Ucchschr., 33, 260 (1951). 21. Brockman, 11., von Falkenhausen, E., Heeff, R., Dorlars, i. and 22. Jensen, 1.. B. and Miller, U. 1., U. 3. Patent No. 2,550,266 (191.8). 23. Gottshall, a. 1., Lucas, 1:. 3., Lickfeldt, Ardeth and Roberts, J. 14., J. cm. Invest., go, 920 (19149). 21‘s m.b0y, Math’ Gottshnll, Re to, ng', Jo Ce m he“, Ea 3., marterly Bulletin of Michigan Agricultural Experiment Station, Michigan State College, East Lansing, Vol. 36, No. I), 1477 (1951,). 25. Hagenstron, 0., fiber den Uirkstoffgghalt unserer heiuschen modems-Men unter besonderer Beruc eichtimg vo_n.g merino: per ora ., es s, Hamburg, l5 . 26. Caseidy, H. 0., Technigue of Organic Cheating, Volume V, Adsogtion and Chromatoggd¥, A. U charger, Ed., In once 11 MI. e, N" ork, e Io; ”SJ-o 27. Kuhn, a. and Uieland, 1., Ber., 11, 962 (191.0). 28. mm, H. w M”, 3., We, a; 73 (19,41)e APPENDICB WK]: CHROMATOGRAPHIC TECWIW’EB W Cassidy (26) gives a comprehensive survey of chromatographic cola-Is, adsorbents and techniques. The choice of adsorbents, eluants and column sise is difficult and is best arrived at on a basis of personal experience. In the present work two types of colurms were used. A straight column reduced to a small diameter affluent outlet as used in elation work. A column sith the lower and comprised of a ball and socket ground glass Joint facilitated extrusion of the ad- sorbent. The flow rate of the oluant was controlled by a stopcock at the effluent outlet. iplug onyrsn glassmolwas inserted into the columnandtapod evenly into the top of the outlet tube. The glasswol plug was then covered uith a filter paper disk which us Just slightly smaller than the inside diameter of the column. This provided an even base for the adsorbent. The column was mounted in a clamp and the outlet stopcock 'las closed. The column us then filled with the initial solvent. ifunneleasinsertedintoacnehclestopperwhichususedtoforaan airtight seal at the top of the column thus forcing the solvent upwards into the tunnel. The adsorbent, uhich had been covered with solvent foratleastanhour, vasaddedtothomnnelandmallowedtosettle by gravity with the occasional assistance of gentle tapping of the 61 eolum no: a rubber mallet. when all of the adsorbent had settled, the stopcock use opened and solvent was allowed to peroolate through the oohnm to aid final pecking. The stopcock was then closed, a second tilterpaper diskmplacedatopoxtheadsorbentand solvent” saintainod over the adsorbent until the sanple was ready to be applied to the column. 0 8 le Thetipot‘athietletubeuasdramouttol m.issidedianeter ndbentatarightangletotheeten. rhethistletnbensthsn insutedintoatwholerubberetopper. amllstopcccksesinserted in the other hole. The stopper us then placed in the top of the column insuchamannerastoproduceanairtightsealandalsoallovthetip otthethistletnbetotouchtheddeottheeolm. meetopeocke at the top and bottom of the calm were opened and solvent we allowed to not until two millimeters or solsent remained above the colon. it thistinethe sample, dissolvedinanninalanocnt or sonnet,” addedthroughthethistletube. Thesalpleusallmdtosettleinto thecolunnontilenlqteoaulinetersremainedabovetheecluln,then a small volume of eolvmt was added through the thistle tube. this againmaneeodtopaseintothecolmontdltnailliaetersrmined abovetheoolm. Mthedevelopingsolvestnsaddedthreughthe thistletubeantiltheliquidlevelreaohedthethistletubetipat tichtinethestopoeokatthetopetthecolmsasolssed. Volatile solventssoohasetherteodedte'vaporlock'andthiasasrenedied bymipulatiesotthestopoeokatthetopetthecolm. 62 gradient Elution Abrupt changes in the type of solvent used as an elusnt often do not produce satisfactory chromatograms. In this research a change of shunt was accomplished by gradually increasing the concentration of the on eluant in the previous eluant. This is defined as gradient elution. motion Collection and Treatment For elution chromatography a Misco fraction collector was used to out fractions. The exact sise of fractions was determined by criteria such as column size and flow rate, which were specific for each type of mixture being resolved. The fractions were taken to dryness by means of a flash evaporator. Special care was taken to keep sample towerature below 35°C when working with unknown materials. The dry sample was dissolved in enlvdrous, peroxide free dieth other and transferred to a 50 ml. tared beaker. The beaker was placed in a vamn desiccator and taken to dryness. Complete drying of sample was indicated by a constant weight. In this manner a weimt-eluant volume graph could be prepared to show the effectiveness of separation. Phyoical and chanical tests, such as infrared spectra and refractive indices, were applied to establish the nature of the sample. The sample he dissolved in a suitable solvent and a portion sent for biomay. The minder of the sample was set aside for further use. All samples were stored at a refrigerated temperature and in sealed containers. In some cases it was necessary w detersine the concentration (w/,,.) of the eluent directly. This was accomplished by pipetting a specific 63 volume of the sample directly into a tared weighing bottle. The couple was then taken to dryness in e gentlelair stream, placed in a 100°C, oven for five minutes, and than veighed. In this manner the weight/ volume concentration ofsolid natainls was determined. APPENDIX II BIOIDGICAL ACTIVITY Iicassaz The smples from Pwus tacamahaca Hill. were tested for activity against Mobacterium tuberculosis, strain 8-37, and Hicrococcus m var. m using the serial dilution method of Gottshall gt _a_l_. (23). gm cricmn RE, lificun sarmles were tested against flcmwcgsge m var. m and zenthgmnas phaseoli by the serial dilution nethod of Gottshall gt. 5;. (23). In all cases, the highest dilution at which growth was inhibited was recorded as the activity of the specific sample . Letivitz Expression and Calculation The specific activity of each angle as expressed as the smallest motor of micrograms (7’) per aniliter of culture broth which inhibited bacterial grO‘ll'th. m: A 1% (v /«r) sample solution was sent for bicassey and he found to inhibit bacterial booth at a dilution of 18512. The con- centration at this dilution is obviously 1/512 of the original suple (mob in this case was 0.01 grams per silliliter) or 19.5 micrograms (7 ) per milliliter. A simple formula for calculating activity in this sooner is given below. e 03:10 Specific activity (in 77ml.) - “‘5'" C8 0 concentration of original sanple in percent (tr/r) D e maxim observed dilution inhibiting bacterial growth For the above example then; " 4 7/211. -9_9_;_}_‘L - 1&9; -19.5 Integretation of Bioaseax Results The bioaesays used in this sork have definite limitations. For sample, solvents such as other and alcohol inhibit the growth of g. tuberculosis at a dilution of lchO and g. m at a dilution of 1:8. Consequently, only dilutions larger than these values can really be considered significant. Also, a small error in the concentration of the sample, shether by weighing or dilution, may cause the end point reading to be shifted one tube higher or loser. a retest of the sample would be necessary to determine the significance of a result in this category. total Activitz In the process of resolving a mixture of active and inactive materials, it see necessary to have some measure of the total activity in order to follos the course of purification. A unit of activity see set up as follows: 1 unit - 1 milligram of solids/milliliter of broth inhibiting bacterial grosth. If a 1:! solution of a sample (10 rap/31.) diluted a thousand times inhibit“ bacterial with, the “I. us “14 to mm 10,” ltd.“ of activity. A fomla for calculating the total activity in a frac- tion is given bales: Total units of activity .. W3 3: n We - weight of total solids in the fraction expressed in milligrams. D a greatest dilution at which a 1% solution of the sample inhibits bacterial growth. M31 A chromatographic sample weighed 0.5000 grams. l 1% solution of this sample inhibited bacterial growth at a dilution of $12. Them Total units of activity :- w, x D . 500 x 512 . 2.56 1 10° 67 APPENDIX III MATERIALS Solvents Diethyl ether obtained from Merck and Company was washed with a solution of ferrous sulfate, dried over calcium chloride and magieeiun sulfate and distilled from over sodium. The solvent ass stored over social in a brown bottle. Merck and Company petroleum ether was redistilled and the fraction boiling froa 30°C to 60°C was used in all chromatographic work. an. ethyl alcohol used in this research was absolute ethyl alcohol obtained from the Comercisl Solvents Corporation. The solvent was not purified further. The chloroform used as Hallinckrodt C. 1’. grade.- The ethyl acetate used in this research nae Merck and 00W o. 1'. grade. mm mumm, moor-20, acme, Activity 1, wasnsed in all ehrosategraphic calms unless otherwise specified. When dis- persedinasaallamntof'atc,thisaluninaproducedanalkaline reacticnintherangecfpnlz. ' Anaeidaluina,pfih.sasasedinpreparingseveralehrosatepans. ItuspreparedinaaannersiailartethatcfxuhnandwielandUfl. Amutyotnooaalnim, cadet-20,803esh,usslurriedwithone nor-a1 hydrochloric acid solution for one hear. The acid an decanted andthealuninawaswasheduthdistilledwateruntilthewsshings reached a pH of 3.9 to LO. It Is very critical that the pH of the washing lie sithin the given range. The alumina was then filtered on alargeBuchnerfunnelandairdriedaiththeainofaheatlamp. The nan-in. no placed in large evaporating dishes and dried a 225% for a period of four hours. Three types of Heels alanine (acid-p3 2, neutral-pH 6, alkaline- pB 10) were obtained non Alupharn Chemicals, Sh G Street, Elnount, L. 1., I. I. ' The activity of alumina is a sensors of the affinity of the adsorbent for the adsorptive am is proportional to the amount of nois- ture in the almina. Brocknan and achodder (28) have established a method for ‘ determining the activity of alnnina based on the relative ease of elution of a series of asobensene dyes by a banana-petroleum ether eluant. In nest cases, the activity of the alumina say be ad- justed by adding water to the alumina. For example, grade I (Brcclcnan) alumina may be changed to grade II (Brochnn) alumina by addition of water in an ancunt equal to three percent of the weight of the alumina. In this research, the activity of all alumina was I , unless otherwise specified. Other aggrbents Corn sta___roh - a commercial product obtained from The Carrier 3W0- Go-p-nv. Len-inc. Michigan. W3 - Hallinckrodt amorous analytical reagent. M - Rasher-0490 vac, Industrial comm Sales, Hes Iork, lies fork. 69 Cellulose «- Solka-Floc, grade Eli-200, Brown 00., Berlin, N. E. 221333 -- Analytical Filter-Aid, Johns-Mannie Compaq. M - Harck and Go., 0. 8. P. grade. silica gel -- Davison PL 100 silica gel, Davison Chemical 00., laltinore, Md. T119319 W ‘ c.2 Dull, G.G. , The investigation of certain antl- - bacterials 116 Mill. and Hypericum prolificum. Ph.D. 1956 . ' Thesis W .2 Dull, G. G. ‘ The investigattlon of certain anti- bacterials 1nPoEgue tacamahaca Mill. and Hypericum prolificmnl ‘ Ph. D. l956______ ;_})a£e"me.i Kama." WWW \l‘HH‘WHW HEW GM 31293 00072 NH