DE2145787A1 - Process for the selection of biochemically active substances - Google Patents

Description

VlB GHEMICAL COMBINATION

Inventor: Dr, Heinz Böhm, Kothen Dr. Werner Kochmann, Bitterfeld.

Prof ρ Dr. Alfred Barth, Halle Prof. Dr. Gottfried Günther, Greifswald Dr ₀ Wilfried Kramer, Wolfen Dr. Hubert Krüger, Bitterfeld Dr, Wilhelm Löttge, Wolfen

Process for the selection of biochemically active substances

The invention relates to a method for uniform Selection of biochemically active substances «,

Bs is known to be the promoting or inhibiting influence chemical substances are examined for biological processes according to very different methods. For example, the processes for pharmaceutical products are completely different from the known ones biological screening tests for herbicides, insecticides, acaricides or fungicides? also is the screening system generally very time-consuming and the margin of error of the mostly rated results is very large «

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2098A0 / 1119

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The previously known selection processes for herbicides not only require a very high level of financial and human resources and time expenditure per test substance, they also have various other deficiencies:

Einmax is; the genetic uniformity of the test material not guaranteed; on the other hand, the test organisms do not develop synchronously and are therefore at the time of contact with the subject to be examined

Substance development-physiologically not exactly comparable with each other »

The evaluation of the tests cannot be mechanized or automated and therefore has a pronounced subjective character,

The conditions under which the tests run are not fully controllable and sufficiently reproducible.
That concerns

- the ecd material required for the test plants,

- the composition of the air surrounding the test material,

- the seasonal fluctuating lighting conditions,

- the rough dosability of the watering per test plant,

- the inevitable fluctuations in the water content of the soil and the surrounding air,

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- the amount of substance used per cm of soil

or leaf area and

- the concentration of the substances to be tested at the place of work.

The purpose of the invention is to eliminate the disadvantages outlined above.
The object was therefore to find a method for the selection of biochemically active substances that allows chemical substances - regardless of their later intended use - to be subjected to a uniform biological test on a suitable object.

This object is achieved according to the invention in that defined amounts of chemical compounds with synchronous or desynchronous liquid cultures of unicellular green algae brought into direct contact and their cell growth or multiplication rate are compared with untreated control cultures under the same growth conditions. As very suitable the unicellular green alga Chlorella pyrenoidosa has proven itself for this purpose.

As a result of the practically unhindered entry and exit of the substance molecules in the algae cells are growth,

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4.04.20 / 1550

Multiplication and, in the case of synchronous cultures, synchronicity as well appropriately stimulated, impaired or tolerated. The comparison of cell growth and / or the growth rate can be determined by physical or physico-chemical measurements, for Example by electronic particle counting, spectral colorimetry or nephelometry. The measurements relate

- on cell growth during part or all of the light phase of the species-specific development cycle of synchronous cultures, _5; .

- on the increase in mass - based on cell growth and cell multiplication - exposed dasynchronous cultures during an exposure period of any length or

- on cell reproduction at the end of the species-specific Development cycle of synchronous algae cultures.

The growth conditions of the algae are determined by the parameters nutrient solution, temperature, gas supply and intensity and duration of exposure are set. The chemical compounds can be in liquid or gaseous form or in solid form are brought into contact with the algae cultures; gaseous substances become more convenient Brought in with the gases necessary to adjust the growth conditions «

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2US78?

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By. Application of the method according to the invention it becomes possible to select a large number of biochemically active compounds in a short time while receiving quantitative information that can be reproduced at any time

In contrast to the well-known inductive tests on higher plants, anatomy and morphology allow it and physiology of the single-row algae, these as a prototype photosynthetically and meristematically more active To consider cells so that the achievable results have a deductive character and allow a fundamental objectification of the test «

As special advantages of the proposed selection, which are generally followed by further tests known methods are to be mentioned:

- It allows the test material to be grown soilless based on defined Mahr solutions in closed Culture vessels on the tightest tree.

- It is based on unicellular organisms that can be counted automatically in every development phase and are measurable and are available in large numbers "

- The test material is genetically uniform and

2098 k D / 1119

£ 143,787

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stable and "with synchronization, physical development

logically same-door. "· ..- ■". ·; - ■■ ·

"* The iesfbedinaungen aind during gesanten 'x ^l ~ est course always controllable and can be, far-

ö ^ lienä autöiaafciacix überacixeri *.

- The methods comn-fe with roohi; geriagea Sut from *
- »The aßgewendetGQ SulDstaozIsronaentrationen constantly · and act directly-on the active ropes

The process of the invention has "special value in the treatment of herbivores; however, it was found that substances of a completely different chemical" type of action, such as insecticide, ikariside, i ^ ua ^ iaid or X ^J haririaka, have an ejectable effect the Qtest organism. so that the inventive features Oelektions- Ahren / jeeiGiiet ersciieint _t allgeaein the -V iaisclier ■ üubstansea on! ferments'l-'ormGntsysteme and Gene au prCif en ·

BATH ORIGINAL 208840/1119

2145737

/] 0

The invention is intended to be closer to readjusting iioiapielaa explained v / ground *

Example Ι »

Freshly hatched 4planospores syni. ? oa cultures the unicellular green alga Chlorella pyrenoidoaa Ohick, Tribe. 211-Sb, become an inorganic liiihrlosang with j? e-BÜ3? A-KQHplex and trace substances so transferred,

that each cm ^ of the suspension is about awei ivilllonen \ & lgen-

aellen contains

A liiter of the oil tariff solution Ii is based on it

don three cable solutions

and B-ausamaont

1000 ml E m. 998 ml &, - + 1 jdI Ii ₂ + Λ ml ll

The 15 years punch ι

Ii ₂ and "BL onthaluen following sub

Art ßubstanaen iiong © Cg) Distilled
Water (1) KH) ₃ . ■■■, ■ 10.111 V. 6.210
2,465 10 Cad ^ O 0.1000

20 9840/1119

BAP ORIÖINAL

- 8th Substances Went-: lot ■ 1QC.1 OsII k.-Jij <2Pi H ₃ BO ₃
MnSO ₄ 'H ₂ O
ZnSO ₄ * 7H ₂ O
OuSO ₄ * 5H ₂ O
Ammonium molybdate 4th 61.0 mg
169.0 mg
287.0 mg
2.5 mg
12.4 mg 04.20 / 1550 ^TV Art FeSO ₄ O 7H ₂ O
Na-EDO) A. 0.69 g
Of95 g Distilled
water * ₂ 11 in 80 ml ko
water
solve and dissolve
Fill up with 100 ml

Each 100 car of the suspension mentioned above is placed in a culture tube, gassed with a mixture of purified air and 1.5% Kbhlendioscyd and exposed at 30 ⁰ O with fluorescent tubes containing red light at a light intensity of about 10,000 lux. The substances to be tested are exposed at the beginning added to the exposure or to the cultures at a later point in time so that solutions or suspensions d, he desired concentration arise} substance-free culture tubes serve as test controls is released in the light of strongly grown mother cells. During the dark phase will

2098A0 / 1 1 1 9

- · - ■ 9 - 4.04.20 / 1550

gassed exclusively with purified I $ x £ t * At the end of this dark phase, the cells in the "treated and untreated cultures are electronically, counted and measured" The results of treated cultures y / erdea compared with those of untreated cultures and thus obtained reliable information on the biochemical activity of the tested substances.

The results of the experiment in the "presence of !!, E-dirnethyl-H ^f -phoessyl urea are shown in Fig. 1" The nine levels correspond to the following concentrations *

Concentration level (%)

Λ ύ 0.1. - BATH ORIGINAL 2 0.05 3 ύ 0.01 4th 0.005 5 4th 0.001 6th a 0.0005 7th Ä 0.0001 8th 0.00005 9 α 0.00001

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21457a?

- 10 - 4Ö

Example room . -'.- ■ - ■ -....- /

The method demonstrated on a substance in Example 1 can also be carried out as a mass test. Since there have been several liquid cultures parasol to one another with different or identical ionaentrationsa the same or differentiated ßub · ^ staaen in Ifealjali: «; gobracM? » Yorbereitung and ^ iizuciifc the ICulturen done analos Example 1. The to be tested Substaasen IÖ three &> Qzeatrauioneai be the culture on added so "that they Raili dsia test object, 0.1 5äig _t 0.01> iig and Q Qqi" gig in jbntairf; occurred. In the Eoutinetsst the restriction occurs; one of these IConaentrationon aöglicii,. '; . M 2y3d.usande v / ird again .. in each ' Tiohse the Z-aiil der Zollen, pro osr SusponsioB "bestiiaMfe or the Uassen- ZVL \ 7aqhB by, i'rilbungn- Τ5ΘΞΐβ κ2Π2Β \ 7Θΐ3Θ spektx-alliolorimetrioohe-liessungeß eriüittslt;! · Bio from aoleitlsA een Verinehrungoraten serve as I5aB for Alcblvität the audited SubotanasD "

The 'ώ? £ έΌηχ30 · 3 one such liasuGatesuS for- 44- Sud- st ans an (Ι ^ Λ) ixa comparison to dea Durcascliaii? Ts-Vermahruarssraton of unhaired Ivoatrolls (0) are in the Ä "bbildun.3oa 2 and 3 entäaibea · DLa iitthzvanzen -i-, 19 * 23 and 27 orwsisen.iUlch himself; in dor IiucügI; ^

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BAD OfHQlNAL

209840/1119

- 11 ~ 4.0 ^

Concentration as biochemically inactive, while substances 7, 13 # 20,24, 32, 37t 39 »40, 41, 4-3 and 44 still have a strong effect even in the lowest concentration · Parallel reads on higher plants confirm this

Example 3 «

The results electronic; *? Cellular data on Älgenkultur on with or without EehandluDg ait 0.001 tigern I ^ -Dimetljyl-2i * -pho! 33rlurea can be seen in Figure 4.

Table 1 zeißfe the Zeliv / aclistuia - expressed in Percent of the i ^ achstuias of the Kbnbroll culture («100 #) under the flow of various at the beginning of the exposure added chemical substances »

Example 41

Figure 3 demonstrates the Xäinf IuB 3- / jjaino- 1t2,4-triazole (iiaitrol) synchronous to the development of green algae} Evaluation is carried out by nephelometry * In Sabeile 2 are the results of such ^{a e} i? Ests for Dubstanaen vornchiedene to-3aE5iaonsestellt ( Exposure function of 8 hours, 3 levels of concentration)

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209840/1119 BAD OR.O.NAU

214578?

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; Example 5 j ^:. . . '' ^: . · '_...-.

The results obtained by spGlcoral & olori-uietric measurements in the ilbsQ ^ tiong area of the main pigment (680 lim) are eiad. Figure 6 ereicht- * lichi verso Medea © ifJoütaentratioaen of 3-amino ~ 1,2,4 "tpisuaol serve as 2es-frsubstanz which iießzeitraum umf t Asst 8 ßtundea · In table 3 are sohließliob. Under- © ueauosen over the Mnf iuJß of three iSbnzentrations different substances listed on green algae cultures ».

209840/1119

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Table 1: Cell growth in the presence of test substances after exposure for 16 hours in comparison to the growth of untreated cultures

Test substances cell growth

£ "in% of the control culture_7

Concentration 0.1% 0.01% 0.001

2-Ohlor- ^, 6 ~ bis-ethylamino-

triazine (simazine) 0 % 0% 0 %

2-chloro-4-ethylamino-6-iso-

propyi-amino-s-triaz iη

(Atrazine) 0 % 5% 15%

2-methylmercapto-4,6-bis-

is opropylami n-s-triazine

(! Prometryn) 0% 0% 0%

N-methyl-ir-methoxy-F * - (4 -

chlorophenyl) urea

(Aresin) 0 % 0% 0%

U- (p-bromophenyl) -W -methyl-

! ^ »- methoxyurea (patorane) 0% 0 % 0 %

N, N-dimethyl-U ¹ -phenyl-

urea (fenuron) 0 % 0% 0%

K, N-dimethyl-N * - / "4-chloro-
-har ns t of I "

(Monuron) 0 % 0 % 0%

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20984 0/1119

4 · O'i ·. 20/1550

Test substances Zellv / aclistum
rf the Äbüfc
ITonaea teatlqa

ό ~

ό, οΐ M.g ~ 0.001

(Diuron)

) iienyl_7 "-Harnsboff (Idnurori)

O % 0

0 %

acid

% 60 SS- '106 %

acid (ITa-Sala) 68

110

2 »4 <* DicIilor« XJh.eno2o ^r ossigsüure (iudnsala)

45 % 108 fo

3-alaino-1,2,4-triaaol

% 16

O-Is opr opyl-plionyi c nr'baiiiat (IPC)

% 52

100 %

O-isopropyl-3-chloroplienyl carbamate (CIPO)

2098Λ0 / 1 1 .- 15 -

Zellvvachüuüxi -; · 3 of the A

· -Tue

pyridyliuTJdichlbrid
(Paraquat?)

23 %

(Diqua-fe)

18 %

Diiaefcbyl-parafchlone

(V / ofa

V / ti

0 %

80 %

2, 2 * 2-tr "iciaorä1; 3ayl) -.

phossplionat

18 f ■ 26

Sulphailharastoff (Suveinil) O

Ohlopcholiaehlorid (OCO)

'r iliresylpliosp

—Hesrachlorocycloliesrah

12 % 34 0 % 32% 80 71 β 92% 95 90 % 97 % _ 100 20 % 25 ^ ■ 93 18 % 62 ^ 100

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0-9840 / 11 9

214578V / f, ft -

received öol ^ .n. '

4. W. 20/1 550 -

- 16
Table 2: Cell growth in the presence of test sub-

punch after 8 hours of exposure in the growth untreated 90%
28% 9 ^ %
90 % Compared to Cultures Test substances Ohlorcholine Chloride (OGC)
Diphenylhydantoin __ cell growth
/ _ in % of iiontrolikuxturJT "
concentration
0.1% 0.01% 0.001% 67 %
O %

Tricresyl phosphate 91% 97% 100%

Suxfanilurea (JäJuvernil) O% O% 30? 6

^) "-Indolylacetic acid (XES) - 85% 91%

Hexamethylphosphoric acid

triamid (Hempa). 90% 96% 99 %

Uranyl acetate 89% 100% 100 %

Dimethyl parathion (Wofatox) 0% 2% 82%

0,0-dimethyl- (1-hydroxy-

2,2,2-trichloroethyl) -

phosphate (Wotexit) 16% 77% 89%

Diphenyl ether 0% 8% 20%

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2098 ^ 0/1110

- 17 - 4th CW-. 20/1550

Test substances cell growth

Z ~ in % of the control culture_7

Concentration 0.1% 0.01% D ", 001%

N-butyl-ir- / ~ 1 ~ (0,0-dipropyl-

phosphonyl) -2-methyl-cyclo-

he: xyl_7-amin 0% 38% 100%

-N- £ ~ 1- (0,0-diiso- "butyl-plio sph. 0 nyl) -cyclohexyl_7 ~ amine 0% 4 % 100 %

3-amino-1,2,4-triazole

(Amitrol) 0% 16 % 45%

2-chloro-4-ethylamino-6-

isopropyl-amino-s-triazine

(Atrazine) 0% 6% 19%

1,1 »-Dimethyl-4,4» -dipyri-

dylium dichloride (paraquat) O% 42 % 74%

2,4-dichloro-ph.eno: xyacetic

acidic 0 % 73% 98%

ChJ oral hydrate 0% 35% 78%

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2U5787

4.04 * 20/1550

Table 3 * Light absorption in the presence of test substances after 8 hours of exposure in comparison to the absorption of the control culture

Test substances

Light absorption at 680 nm
~~% of the absorption of the

concentration
0.1% 0.01% 0.001%

3-amino-1,2,4-triazole (Amitrol)

oL -hexachlorocyclohexane f -hexachlorocyclohexane

%

%

%

0,0-dimethyldithiophosphoryl ~ acetic acid-N-methylamide 10%

Tributyl tin oxide 30 %

Isopropyl 4,4 · dibromobenzilate (JSeoron)

Triphenyltin acetate (Brestan)

Dichlorodiphe nyltrichlorät Han (DDiE) 12%

1-maphthyl-li-iiiefchylcarbamate (carbaryl) 9 %

Potassium nitrate

100 %

8 _f 4 % 21.0

74.0 % 82.0%

31.5% 100.0 %

76.0 % 100.0%

41.0 % 65.0%

40.0 %

56.0

35.0% 56.0%

33.0% 100.0%

100.0% 100.0% - 19 -

4.04.20 / 1550

Test substances

Light absorption at 680 by n % of the absorption of the

concentration
0.1% 0.01% 0.001%

Matriumdilayärogenphospiiat 100%%

100 %

4,6-dinitro-o-cresol (Hedolite = DHOC)

0 %

%

4-chloroplienyl-2 _f 4 _f 5-trichlorophenyl sulfide (0? Etrasul)

100

100

N-1 -Haphthyi-phthalic acid (Haptalam)

%

108 %

1,1-bis -> (- chlorophenyl) -2,2,2-trichloroethanol (Milbol)

%

? 098AQ / 111Ö - 20 -

Tabel

2U5787

- 20 - 4.04.20 / 1550 Substances with low plant activity. applied in a 5x10 percent solution

Substance Percentage absorption at ram compared to, for untreated control culture

EOL MgSO

H ₃ BO ₃ NaOl

OaCl ₂ Na ₂ GO ₃

MnOl ₂ FeOl ₂ MgOl ₂ Na ₃ PO ₄

10H ₂ O

Ga (N0 ₃ )

^3-Amino-4-f äthylbenzyl-4-chloro-2 * -äthylphenyläther 84.5 102.0 99.0 98 90.5 ü

103.0

93.5

90.0

110.0

81, ü

98.0

103.0

100.0

107.0

97.0

80.0

5-chloro-2-hydro2? Y-3-ethylpropioph.enone 70.4

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2 (398 ^ 0/1119

214578?

4, ü'i. 20/1 31 ■ · '■>

substance

Percentage absorption at 6ΰΟ nm in comparison to the non-shuddered Control culture

acetylpiienoiiy) -ät hosyJT ^- -diäthylflther

-1,2,5 _f hydrobenaoic acid ethyl ester

n-butanol

Oleyinatiiyltraurin-Ka Suprasil

Ca-alkyl-arylcv.lf onate 60

(Tekunil CA 60)

(aliphatic «IColilenvjasserstoff)

Dispergator BO
(oxethylated j

71 f β #

104.0 %

109.6 w

92.0 %

70.4 #

Untreated control room 77 _t 3% 93.5 years

106.7 % 100.0%

2 0 9 8 A 0/1 1 1 BAD ORIGINAL

Claims (1)

J? a t ο η t a η 's ρ r ü c Ii e s. Iy method of selection biooheaisca v / irks aia , daduroli ^ ejccnnzgiGlmoop that def i ~ Mengea Gheiaischer · "VerTbiudun ^ en mit; synchronen or dosynclironen ilüssiskoltsloiltur-ea one-inch green algae in uEunitL-el "bareQ iContaki? gobraGhfc and their Zollwachsiroa or Veriaehrungs— rat © with untreated IiontrolllailturGn under The same requirements must be met. 2 # The method according to claim 1 "dadurcli r; ekerinzoiclmet ^ that Chlorella pyrenoidosa is used as a single green algae" 3. · AV Ahren according to claim 1 and 2 characterized ₉ b geichnet "that the comparison of the S and / odex ^ the Veriaehrungsrate by physical or physico-cheaische LiGssun ^ s * is carried out 4-jt method according to claims 1 to 3 " characterized " that the measurement is carried out by electronic particle counting "Spoktralkoloriraetrie or ITephe loiaetrie" · "- 23 - BATH ORIGINAL 209 8-0 / 1119 214578 ' - 23 - 4.04.20 / 1550 5. The method according to claim 1 to 4, characterized »that the chemical compounds are brought into contact with the liquid, gaseous or solid form with the Ji'lüssigkeitskulturen. 6. The method according to claim 1 to 5 » characterized 1 that the growth conditions are set by the parameters nutrient solution, temperature, gas supply, intensity and duration of the exposure. 7. Method according to claims 1 to 6, characterized in that gaseous substances are introduced into the liquid cultures with the gases required to set the growth conditions. 8. The method according to claim 1 to 7 # characterized « that several liquid cultures are brought into contact in parallel with one another with the same or different concentrations of a substance. - 24 - ? 098 £ 0/1119 - 24 - 4 * 04-. 20/1550 9. Procedure according to. Claims 1 to 8, characterized in that "several liquid cultures are brought into contact in parallel with one another with the same or different concentrations of different substances" Brooch Blank page