Doc 0000163370

. !··;...'· r' • ' ' '~ • -- • ~"' ... ~~:;~:'-v. :~-.::.;.-;.l,.; -~" • ~ • :. .I:~ ..... ,. .1 ! t i I ! COPY--~~--OF--~4~· __C OPIES I - i I are five copies of the i ' .,,...:aA.,,,t contract, \ and covers t.~e \ , er 31, 1969. ~he report includes I a statement of expenditure of funds and c~~pletion of I I the assigned task. ) - t .·. .. Pleasa let us know if ~~ere are any questions or c~~~nts as to its contents. Sincerely· yours, ) ., -~----·--· THIRD QUARTERLY REPORT ABSTRACT Areas of activity during the third quarter are listed in this paragraph and elaborated below. Progress continued on an accelerated level on the basic toxicity and be havioral screening and additionally included programs~ cardiovascular pharmacodynamic testing of a group of compounds and isolation and testing of extracts of several - oriental plant products. A total of ... .... 5~3- compounds wer . e received for screening during this In time·interval~ ~ addition, a natural products chemist began work on preparation of extracts of oriental plants reportedly of medicinal value according to Chinese folkloreo Several of· . these plant extracts have been tested for biological activity and toxicity in mice; work has proceeded to the point of separation of several active fractions. The visual discrimination apparatus has been received fro~ and assembled. · To date 61 per cent of the funds have expended and 61 per cent of the task completed. bee~ - 2 - PRIMARY SCREENING During the current reporting period, 36 compounds were tested in mice by the acute toxicity screen and locomotor activity Practically all of these compounds showed tests~ a s~itable minimum safety ratio between the LD and MED 50 50 to warrant further testing. Five compounds showed out standing safety ratios. (1000 or greater) These were ·o and In ·addition, 10 compounds showed ratios between 100 and 1000. all compounds were tested in cats at a dose (mg/kg) ~owever, 1/10. the mouse LD level, for the effect on physicai, neuro~ 50 logical, and behavioral statuso Ten of these compounds ·-.) shc:>wed _significant at:tivity. -~- . . . . ;.- ... ' .. ..._. ., ~ --' SECONDARY AND ADVANCED BEHAVIORAL SCREENING Thirty-four compounds were evaluated in hooded rats by the motivation and sequential response behavioral tests. The dosage (mg/kg} administered was 1/20 the mouse LD , The page contains a stylized graphic on the left depicting a large, mechanical vault door with intricate gears and dials. To the right of the vault, white text is displayed against a black background. The text describes "The Black Vault," an online database of declassified government documents, specifically mentioning the MKULTRA/Mind Control Collection and providing a website address. There are no photographs, handwritten annotations, official stamps beyond the text itself, forms, diagrams, tables, redactions, or visual evidence of experimental procedures. of these compounds ·-.) shc:>wed _significant at:tivity. -~- . . . . ;.- ... ' .. ..._. ., ~ --' SECONDARY AND ADVANCED BEHAVIORAL SCREENING Thirty-four compounds were evaluated in hooded rats by the motivation and sequential response behavioral tests. The dosage (mg/kg} administered was 1/20 the mouse LD , 50 or lower -if overt toxic signs were evident in preliminary test rats. In both the motivation and the SRBA tests a number of compounds showed some psychopharmacologic activity, as evidenced by an increase or decrease in start or run speeds or a decrease in the number of rewards and an increase in the percentage of errors. For further eval uation, test compounds were administered to monkeys, and the physical, neurological, and behavioral effects, including changes in shuttle order behavior in the shuttle box test, were deterrninedo This is further described in detail in a separate section Sixteen compounds were studied in below~ monkeys. - "\, ., .• ·~· PHARMACODYNAMIC SCREENlNG Pharmacodynamic screening c3zrie1 out on compounds w~s 1~ in anesthetized cats.. I'he p r.t"'t:>c:>.l t::>L the p.to,;oedu.r~ and the results obtained a.:::e d-2s::r .tbed bel~·Wo Pharmacodynamic S=reenin£ in Anes~het1zsd Ca~s Compounds·were for ph3!macodynami= activity in screene~ cats anesthetized with cC-chloral.:>se (80 mg/kg, i.p.). blood pressure, heart r3te. respiratory movementsJ Arteria~ and were recorded on. an E and _Effects EKG~s 1>1 Physiogr?P~·- on responses to the 3dminJ.st.rat:i -) and to vaoal and to ... st~ulation . carotid artery or-elusion were measured. . Challenging drugs were usually administered at five·m.inute intervals or after arterial pressure had t:o th; control level. All ret·.lro~d injections were made via polyethylene cannuls inserted into the femoral vein. Each compound was usual~i tested in one an~mal~ The initial dose was either 0.3 or 1.0 mg/kg. doses were in Subsequ~nt creased or decreased by o.l'le-·half of a 1ogar ithmic in~erval1 depending on whether a response was eli=ited~ The results of these are in the expe.ri~ents surr.marli~d Appendtx. activicy observed w1th 15 of the Intr~nsic w~s 18 compounds tested.. One cornpouo:ld. caiJsed an increase in blood pressure and 12 compounds cacsed de~:eased blood j • i~~~-;~-:?:~~{~~' : ~., - 4 - pressure... l'he duration of each of the blood pl:·essure changes was less than five minutes.. EKG ~.tol.tage increases were observed after a1rntnistration of None of the compounds affected the responses to any of the challeng ing drugs or pro:edares .. BEHAVIORAL Tl!'Sl'T~G OF· SQtTI.RREL MONKEYS Certain personnel The image displays a typed document with several redactions. A date, "February 18, 1970," is visible at the top. Below this, a line indicates "COPY 2 OF 6 COPIES." The main body of the text discusses enclosed quarterly progress reports for a contract covering October 1, 1969, to December 31, 1969. There are several black ink redactions across the page, obscuring parts of the text and what appear to be signatures or names. A handwritten annotation of "176" is circled in the bottom right corner. The page also shows faint vertical lines that may indicate a margin or binding. The page features a standard typewritten report with a bolded "THIRD QUARTERLY REPORT" and "ABSTRACT" heading. Along the top right edge, a handwritten annotation reads "COPY 2 OF 6CC", suggesting it is a duplicated copy of a document. There are several prominent black redactions on the page, one obscuring content in the top margin, another partially obscuring text in the abstract's paragraph, and a large, dense black rectangle at the bottom of the page. A small, curved handwritten mark is present on the left margin, and a small circular stamp-like mark is near the top center. The overall impression is of a formal report with some administrative markings and intentional obscuring of information. - 4 - pressure... l'he duration of each of the blood pl:·essure changes was less than five minutes.. EKG ~.tol.tage increases were observed after a1rntnistration of None of the compounds affected the responses to any of the challeng ing drugs or pro:edares .. BEHAVIORAL Tl!'Sl'T~G OF· SQtTI.RREL MONKEYS Certain personnel a1.di~.l.cns., pr~du.ral ·implementa-:..Lcns and 1 eq'..li.pment modifl.c.at ions were i:1sti to te -c.he tu~ed fac~lita operation of the r-eel testing pr·ogram .. s~ui monk~y Per so nne 1 Addi t i:-ns - A behav icral scientis·t. was added to ··- ,.. - the staff and asstgned to the squoirrel monkey testing procedureo . 1i"t'.-~- -.-~.... ~·z•J:t~··• ·-·~~~~~-:1:'~~- ·$:a-~=--· · )·· __-· .~-:· ·-~ipm · 'e'nt-· MO,:::!,r-T'Zi!~;~.·v~··~"·'""l:fC1':::r-:l·~~··au~;t~··.;-·.~;1:,1.":15. . · ~.ov"": · t.n-e:~~"-l" ., - ,. - . · _ " _ '" . .:_ ' .... . . ,"""-.."-~ ef f l.C ie~cy of the test.i!i.; proced·:l.!"es and to insu:.:e the val1d1.ty of are listed below: r~su.!ts ob~5:ned a" The tes-:in~ ar~:~ contain1.ng the shuttle box was isolated from the area and the by data~rcecord.J.ng invest~gator a boo~:h made of sheets of opaque black plas~ ic o Both th9 lnvestigator~s and ~esting areas wer: then slmlla dy separated f.::om ths g~neral room en~Tiron- rr.~='\., Thus) -:he room l:.;h:s could remain lit dur~ng testi~g procedures so that the untreated monkeys wo~ld be e~posed ~o a normal light-dark cycleo b... A one·way mtrroi was placed between the acea of t:he ~.nvest 1g~r::-r and that of the tes~ subject,. so that the monke-ys pP.~formed thel.I tasks while being v1sually 1solated from the influence of the 1n ·) • vestiga .. or 01= g~r.~ral z~orn movement.., but fully . ob*erved by tho in~es~1ga~o~~ •· 5 - c.. General lightJ.ng was placed in the area fl:Jo:r~scent of the s, bu~tle apparatus to prov1de an ov~rall 11- luminance so the monkeys could readily see the visual cues and the investigator could easily the de~ermine roonkeysu 1dent1f1cation and behavior patternso de A mechan1.cal door closing system for the shuttle boxe.s added so that the entire testing operation., w~s after the monkey was placed in the shuttle boxu re mained isolated fz:om the monkeys.. This ~·isually further intra-test disturbances and vari reduce~ ability., ·:) The modifications were designed to isolate as .. . abo~e . .. ~ much a s~~c.ss.;i.b~s;t.,ani.maJ.. . .,~. .. ..a..nv-~"E--~~~,~~~. .~ "'· • situa1:ions., both visual and auditoryo This is essential fot the propagation of The page contains a typed report with two sections: "PRIMARY SCREENING" and "SECONDARY AND ADVANCED BEHAVIORAL SCREENING." There are several dark, horizontal redactions at the top of the page, obscuring some content. There is also a handwritten annotation that appears to be a page number "-2-" in the upper right quadrant. The text discusses the testing of compounds on mice, cats, rats, and monkeys, with a focus on toxicity and behavioral effects. There is no visual evidence of experimental procedures, equipment, facilities, or any photographs or diagrams present on this page. mained isolated fz:om the monkeys.. This ~·isually further intra-test disturbances and vari reduce~ ability., ·:) The modifications were designed to isolate as .. . abo~e . .. ~ much a s~~c.ss.;i.b~s;t.,ani.maJ.. . .,~. .. ..a..nv-~"E--~~~,~~~. .~ "'· • situa1:ions., both visual and auditoryo This is essential fot the propagation of motivational data and response cons~stencyo e.. A styrcfo~.m weighing cage and a plexiglass injection · t11be were constructed to reduce mechani-:al injuries· due ':.o handling; contact and infection from abrasiono 1 . --- Procedural Chanass - Certain changes were made to maximize tne _...__.. data preeently and to provide for an increase in data ob~a~ned in the future .. Scheduled tralnJ.ng sessions were· initJ.ated., · These·· will a~·· continue until the full complement of trained proje~ted monkey gro,Jps is attainedo At present.1 one shut.tle group of monkeys crained to both a positive and negative .) thre~ • • - 6 - reinforcement schedule and one group of three trained to a regime are available for group shuttle testing. nega~ive In additionv seven males, which can be trained on a positive reinforcement schedule to replace any established monkeys or which can be used for the visual discrimination te.s ting programu are.in the colony~ bo Seven female squirrel monkeys were purchased and condi tioned to the shuttle box apparatuso These are used daily to determine a dosage level of test compound (mg/kg) which produces minimal behavioral effects and can thus be applied in a shuttle operationo These monkeys are important in the detection of behavioral effects observed in the shuttle apparatus or in the home 9 cage, caused by varying drug dosage levels.. The procedure on individuals includes the observation of many behavior '~ tterzf'ri and motiv ~efonif~-sta~d'S'~rid4fivftt''*mtfclf:l?.':!fif~:<r.::.~~*''l\i':t. . ,.,.X!>-;:-:·~ mation about a specific drug effect which may be present • . (l.) Observational Procedure and Criteria. The individual is observed and subjected to the stimuli presentations every five minutes in the shuttle box for a 30 minute control period-prior to dosing. The first dose is injeqted, and the animal is placed back in the shuttle box and observed for one hour. Subsequent increasing dose levels proceed hourly until the maximum determined dose is reached '(cumulatively). The individual is then observed in its home cage after four hours and after 24 hours~ The behavioral activities being cataloged during this procedure are: (1) reactions to observation and handling by the investigator (flight, attack; The visible content of the page is primarily text from a declassified document, likely a research report. There are no photographs, stamps, forms, or diagrams present. However, there are several black ink redactions obscuring portions of the text, most notably a large block across the middle section of the page. There are also a few small handwritten marks, including what appears to be a small "3" and a hyphen to its left, positioned near the top right of the page. The text itself describes "Pharmacodynamic Screening" and details experimental procedures involving anesthetized cats, blood pressure measurement, and drug administration. Subsequent increasing dose levels proceed hourly until the maximum determined dose is reached '(cumulatively). The individual is then observed in its home cage after four hours and after 24 hours~ The behavioral activities being cataloged during this procedure are: (1) reactions to observation and handling by the investigator (flight, attack; or passiveness); (2} grasping ability (to bars and manipulanda)J (3) feeding behavior (eating or not) 1 (4) reaction to blinking .light to mild shock - 7 - (6) reaction to sound of buzzer (alert, orientation, etcg); (7) shuttle response (ability to shuttle after light cue; door open, and shock if. used); (i) level of self-manipulative behavior (grooming, self-inspection, etc~); (9) locomotor coordination, speed, ability (natural under stimulation and during shuttlei; 1 (10) level of general activity; (11) amount of sleep; (12) behavior during crouching (alertness, head orientation responses, eye fixation) a {2) Ipjection Procedure. All injections of test compounds are given intravenously via a caudal veino The total dose level is determined by extrapolation using as a base the lower limit of the 95 per cent confidence limits of the LD for mice. The dose to monkeys was 50 calculated using the conversion formula of Paget and ~'"'~Barne-s1 which is based~ on surfac·e- area1at'so-oy _,_-,,.,,., .., _., -· .... .;... .. --,;;..,..._~. reference to Dro Elton Homan's equivalent surface area 2 dosage conversion factor card as repor~ed by Freireich • The maximum dose level (mg/kg) administered to a squirrel monkey as extrapolated from the dose to a mouse to yield an equivalent dose based on surface_ area is determined by multiplying the mouse dose (mgjkg) by a factor (Oo45)o Aliquots of this total dose are then given in one hour intervals until effects are observed or the extrapolated dose level is reached. REFERENCES ro Page£; GwEo~ and Barnes, J~Mo, 1964~ Chap~ 6, Toxicity Tests in Evaluation of Drug Activities, Pharmacometrics, P .. R. . Laurence and A. . L. Bacharach (edso) •. Vola .1, Aca~emic ····· Press, pp. 135-167. 2. Freireichi E.G .. et al., 1966o Quantitative Comparison of 7 Toxicity of Anti-cancer Agents in MouseJ Rat, Dog, Monkey, .. and Man. Cancer Chemotherapy Reports, 50(4), 219-244 .. - 8 ~ NATURAL PRODUCTS Chinese Toxic Compounds: The names of 47 intact plants, 1~ roots, tubers, and flowers were selected from a medieval Chinese medical and pharmaceutical reference (Li Shih-Chen, 1551) .. Botanical names of these plants are as follows: 1 The document is a typewritten page with some handwritten annotations and redactions. There is a handwritten number "4" at the top center, likely a page number. There are two black marker redactions in the top margin that obscure text. The text discusses behavioral testing of squirrel monkeys, including personnel and equipment modifications for the testing procedures. Bullet points "a" and "b" describe specific details of the testing area setup and the use of a one-way mirror. There are no photographs, stamps, forms, or diagrams visible on this page. and Man. Cancer Chemotherapy Reports, 50(4), 219-244 .. - 8 ~ NATURAL PRODUCTS Chinese Toxic Compounds: The names of 47 intact plants, 1~ roots, tubers, and flowers were selected from a medieval Chinese medical and pharmaceutical reference (Li Shih-Chen, 1551) .. Botanical names of these plants are as follows: 1 .. Rheum officinale 2 .. Phytolacca esculenta 3 .. Peucedanum Japonicum Thunb .. 4 .. Potentille maxim cryptotaern~ae 5 .. Euphorbia adenochlora Morr et Dene 6 .. iuphorbia sielaldiana morren et Decaisne 7 .. Euphorbia heliscopia L. a .. Galarhoeus siebaldianus Hara .. 9 .. Euphordiae Lathyris Seme~ 10 .. Hyoscyamus agrestis Kitail 11 .. Unjitsu (unknown in Western terminology) 12 .. Richinus communis L.. , 13 .. Orixa japonica (Dichroa febrifuga) ~hunb.. -14 .. Andropog,on.. ~g.hum_.,S~a.t . .... __ " 'l,A.;:.,a...,__y_uJ.gftr_i~ _ H ~ck -:';;~ 15 .. Veratrum nigrum L .. 16 .. Leucothoe Grayana Maxim 17 .. Aconitum Fischri Reich 18c. Tenyuh.. {unknown in Western terminology but similar to Aconitum Fischori) 19 .. Aconitum Chinese Sieb .. (daughter root)· 20 .. Aconitum Chinese Sieb (small root) 2lw Aconitum Sinesis 22 .. Jatropha Janipha 23 .. Rhizoma Arisaematis 24 .. Arisaema ringeus Schott varp Sieboldi Engl .. 25 .. Hydrosme Rivieri Englv 26 .. Pinellia Tuberifera 27 .. Polygonum bistorta L .. 28 .. Podophyllum versipella Hee .. 29 .. Pardanthus sinensis 30 .. Iris tectorurn Maxim 31 .. Hosta Sieboldiana Engl. 32 .. Kaempferris Galanga 33 .. 'lachoso (not in We5tern terminology) 34 .. Stramonium Datura Alba 35 .. Rhododendron Sinense Sa .. s .. z .. 36 .. Daphne Genkwa et. - 9 - Wikstroemia Japonica Miqw 37~ 38. Baddlea Japonica Hemsl. 39.. Illicium anisatum L. Shimmia Japonica Thunb. 40~ 41.. Ranunculus sceleratus L. 42. Ranunculus acer. L. var .. Japohicus Maxim. 43.. Aconitum Lycoctomum L. s. z. . 44.. Urtica Thunbergiana et 45.. Gleditschia glauca HK 46w Alocasia Macrorhiza Schott 47. Rhus Toxicodendron L. var. Radicans Miq. Plants numbered 4, 13;. 17, 22, 28, 29, 30, 31, 34, 35, 37, 38, 42, 43, and 44 have been checked by the Natural Product Section of the National Cancer Institute and were negative in anti-tumor testso However, several fruit-bearing Euphorbia species showed activity against Sarcoma 180, Walker 256 and Lewis lung carcinoma .. 1 Several of the active principles are in the process of fractionation, but in only one case was the active agent isolated and this was The page contains typed text describing experimental procedures and modifications. There are no photographs, diagrams, or forms. There is a circled minus sign followed by the number 5, likely indicating a page number. There is a handwritten symbol resembling a checkmark within a circle on the left margin. There is a heavily redacted black rectangular box at the bottom of the page, obscuring what may have been text or an image. The overall appearance is that of a research document with some internal markings. Cancer Institute and were negative in anti-tumor testso However, several fruit-bearing Euphorbia species showed activity against Sarcoma 180, Walker 256 and Lewis lung carcinoma .. 1 Several of the active principles are in the process of fractionation, but in only one case was the active agent isolated and this was repor~ to_ ,.~e ta!lA,~n.. . .. o;chroa;.-..~~:tJr:i~\lS:~.:::i~~,'!!!,.;;.,.lli*'!~-a.!lllJ:tl~.~"'~~.;,;,;;.;.· KB cell culture.. This plant is being fractionatedg but the Ci_Ctlve agent has not yet been isolated.. The rhizome of one species of Arisaema was active aginst Lewis lung carcinoma, but the active compound has not been identified. Twenty-four species selected from the above list of materials were ordered from a Chinese pharmaceutical Because the 1_ng some delay in acquisition, these· 24 species also have been ordered from other sources in ln addition to the plant materials described above a Chinese 9 pharmaceutica}. -and extracts of black t~_(i, gr.e~n tea, and aronia fru1t were tested for toxicity in mice .. - 10 - Some a Chinese pharmaceutical, was The chemical composition of the material is unknown. Ten milliliters of distilled w..a ter was thoroughly mixed with 1~125 grams of this compound, and the material was injected in mice~ No effect was observed. In the appended comp~ter printout tables, this material is reported as No~ 300000. The tea extracts showed some activity in mice; however, it is likely that the effects of caffeine masked any pharmaco logic action by other ingredients. The tea extracts are designated in the appended computer printout as follows: Extracts of green tea: Nos. 110000, 120000, and 130000 Extracts of black tea: Nos. 140000 and 150000. Fruits of the aronia plant {Malus Halliana Koehne) were harvested during the winter from trees located after all the leaves had falle~ and only brownish red fruits remained. The fruits were immed iately frozen and stored at. -15° with dry ice in a styrofoam cabinet~ Frozen fruits {32.51 grams) were blended first and_ then homogenated with an electric blender for 10 minutes and extracted twice with 100 milliliters of distilled water by stirring under nitrogen for two hours at room temperature~ The filtrates were combined and concentrated to dryness using a freeze-drying procedure. The residue of the filtrate was re extracted twice with 100 per cent acetone {100 milliliters) • The acetone extracts were combined and evaporated to dryness under a vacuum. • - ll -· The residues from the water extract fraction and The document is a typed page titled "- 6 -" at the top. It appears to be a research or experimental report, detailing procedures involving squirrel monkeys. The text describes conditioning animals, dosage levels, and behavioral observations. There are no photographs, stamps, forms, diagrams, tables, or redactions visible on this page. The visual content is exclusively textual information presented in a standard document format. combined and concentrated to dryness using a freeze-drying procedure. The residue of the filtrate was re extracted twice with 100 per cent acetone {100 milliliters) • The acetone extracts were combined and evaporated to dryness under a vacuum. • - ll -· The residues from the water extract fraction and the acetone extract fraction were each made up to a volume of 32 milli liters with distilled water. Each milliliter contained the extractable material equivalent to one gram of freshly weighed fruit. Aliquots (40 ml/kg, ml/kg, and 5 ml/kg) of these 1~ solutions were injected intravenously in Dublin (DUB/ICR strain) albino mice via a tail vein. The effects of the injections are presented in the computer assembled tables (fraction numbers 220000 - acetone, 210000 - water) and show that an active compound was soluble in acetone. The mice died during, or immediately .after intravenous injection of a dose of 16 ml/kg of the acetone fraction. The previous experiments showed that at least one toxic compound was in the acetone fraction, so the compound(s) was dis~ributed (wereJ further separated andpurified with a series of organic solvents since the appearance of the acetone soluble material(s) in a particular solvent fraction would provide information concerning the polarity and chemical properties of the compound(s)c Another quan-city (61.23'grams) of fruits was blended with an electric blender and extracted twice with 200-milliliter aliquots of n-hexane {dielectric constant 1.9), chloroform (4.,8), diethyl ether acetone (20.7), ethyl alcohol (24.3), and distilled (4~3); water The extractions were carried out at room temperature. (78~5). Insoluble material was filtered from the extraction fractions using Whatman No" 1 paper. Weight Per Cent of Biological Fraction Fresh Activit:t Wei~ht ~ n-hexane 0.322 0.52 chlorofot"m 0 .. 521 0.85 ++ ethyl eth~r 1.157 1.88 ·-~. --···· ·- . - acetone-! 0.456 0.74 acetone-2 5.668 9.80 +++ ethyl alcohol 2.0917 3.41 distl.lled water 1.331 2.17 +· - 12 - As shown above, least two active compounds were extracted a~ from the aronia fruits. The designations "acetone-1" and "acet::me-2" indicate different steps of fractionation: the residue following the diethyl "ether extraction was extr-acted t:\'lice with 200 milliliters of 100 per cent actone. The acetone ext:racts were filtered and the filtrate was to dry evap~rated ness. The sap of the fruit is miscible in acetone, and there fore it was possible to extract a polar compound in the acetone. The residue was taken once completely to dryness milliliters of 100 per cent actone. The acetone ext:racts were filtered and the filtrate was to dry evap~rated ness. The sap of the fruit is miscible in acetone, and there fore it was possible to extract a polar compound in the acetone. The residue was taken once completely to dryness in a desiccator and then re-extracted with acetone (acetone-1). The residue of the acetone extract was freely soluble in water !acetone 2). One-tenth aliquots of each of the residues from each extract were dissolved in 6.2 milliliters of distilled water from which port.Lons (40 rnl/kg, 16 ml/kg, or 5 ml/kg) were injected in ·-···-··m· -·J-.~c-~e-· -~- --· -Nonpolar sol vent extract fractions were suspended -in - 0.5 per cent methylcellulose and injected in the same volume as the polar fractions. The nonpolar fractions were n-hexane, . . chloroform, diethyl ether, and acetone-1. The water fraction contained an active compound which resembles the active comp~und 1n the acetone-2 fraction. The results of biological testing are shown J.n the appended computer prinout tables. The acetone-2 extract is.No. 214200 and the water extract is No. 21600. At. least two biologically active compounds were from se·;;ara~ed the aronia fruits by the described procedures. One nonpolar ~s and the other J.S a polar c~mpound. No difference in biological was observed after the pH of the extracts was adjusted act~~'ity - 13 - with sodium ·hydroxide to 6. 8 from 2. _5 - 3. 0 in the acetone, alcohol, or water fractions. e~hyl The active compound in the acetone-2 fraction was purified further. Four hundred sixty-nine milligrams of the acetone-2 fraction residue was dissolved· in 0.5 milliliter of dis.t illed The water solution was streaked onto six TLC plates, wa~er. which were Brinkman precoated and had a 0.25 mm thickness of Silica Gel-G containing caso as a binder. The TLC plates 4 were developed at room with an ethanol (96 per ~emperature cent):water:ammonia solution (23 per cent), 100:12:16 v;v, i. solvent system {"Braum and Genneu, 1962 ·, Chrornatg. 7, 56). Chromatographic separation continued. for·eight.hours. The plate was divided into 10 parts on the basis of Rf values. Each part was scraped off with a spatula and suspended in ---~-----~-.di.stilled...wa:t:~~-~be_._water extracts were lyophilized and • ~ .•. - ·- .,: • - .:· ......_ :;:'::'~,~=-=::;-:-·~ ..~ .- the residue was dissolved in 3.1 milliliters (two times con- centrated solution of 6.2 milliliters*) from which portions were used This declassified CIA document page contains typed text detailing experimental procedures and references. There are no photographs, handwritten annotations, signatures, stamps, forms, diagrams, schematics, organizational charts, or tables visible on this page. However, there is a single, small black redaction in the bottom right corner of the page, obscuring some content. The main visual element is the typed text itself, which describes a scientific protocol for drug administration and measurement of effects in laboratory animals. values. Each part was scraped off with a spatula and suspended in ---~-----~-.di.stilled...wa:t:~~-~be_._water extracts were lyophilized and • ~ .•. - ·- .,: • - .:· ......_ :;:'::'~,~=-=::;-:-·~ ..~ .- the residue was dissolved in 3.1 milliliters (two times con- centrated solution of 6.2 milliliters*) from which portions were used in the bioassay. The Separation of Active Compound of Acetone-2 Fraction Using Thin-Layer Chromatography Weight of Material Weight Biological Frac~ion No.1 With Binder of Binder Activit:t: l 167.1 29.8 -+ 2 132.2 29.8 3 126.0 29.8 .,..,. 4 79.7 29.8 5 47.2 29.8 + .. 6 45.8 29.8 7 45.4 29.8' 8 48.8 29.8 9 47.6 29.8 10 45.0 29.8 TOTAL 784.8 298.0 l Correspond to Rf values. Recovery of experiment procedure was . (784 • 8 - 298 ..4 86. 8 486.8/569 X 100 = 85.5% · * nn ,..,.; n:=tl wo'i ah+ nf th~ mater~a£ was4 569 mcr from 6 -2 a of fresh we~aht. - 14 - The active compounds located in the acetone-2 fraction have no~ been completely purified yet, so they have not been identified. This will be done in the future, as will the purification of the active compound which is soluble in chlorofor:m • . The aronia extracts which were. active, showed an interesting suggestive of that produced by several types act~vity, ~f compounds, such as stimulants, morphine-like drugs, or hallucinatory agents. GOALS FOR NEXT PERIOD Work will continue on all aspects of the behavioral screening as described in this report. Additional compounds will be obtained .for testing. Training of monkeys in the visual discrimination will be initiated. Natural product isolation ~ask will be continued and extracts tested. Pharmacodynamic screening in anes·thetized cats will also be continued. .. . February 13, 1970 Submit~ed: The document page is primarily text-based, featuring a numbered list of plant names under the heading "NATURAL PRODUCTS." A small section above the list indicates that the information pertains to "Chinese Toxic Compounds." There are no photographs, diagrams, or forms visible. Notably, there are several black marker redactions obscuring portions of the bottom edge of the document, suggesting that some content has been deliberately removed. The page is numbered "8" at the top center. The document is a typed pa