1982, Vol. 45, pp. 31-34, © Hippokrates Verlag GmbH Jouralot Planta Medicinal medica Plant Research Brine Shrimp: A Convenient General Bioassay for Active Plant Constituents B.N. Meyer, N. R. Ferrigni*, J. E. Putnam, L. B. Jacobsen, D. E. Nichols and J. L. McLaughlin Department af Mecicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, and oll Cuture Laboratory, Purdue Cancer Center, Purdue University, West Lafayette, IN 47907 Received: January 10, 1982 Key Word Index: Artemia salina; Brine shrimp; Bioassay methods; Euphorbiaceae seeds; 9KB and 9PS cytotoxicities. Abstract A method, utilizing brine shrimp (Artemia salina Leac), is proposed as a simple bioassay for natural product research, The procedure determines LCs) values in ug/ml of active compounds and extracts in the brine medium. Activities of a broad range of known active compounds are manifested as toxicity to the shrimp. Screening results with seed extracts of 41 species of Euphorbiaceae were compared with 9KB and 9PS cytotoxicities. The method is rapid, re- liable, inexpensive, and convenient as an in-house general bioassay tool. Introduction Many new natural compounds are isolated, cha- racterized, and published without any biological te~ sting whatsoever. Their useful biological activities can remain unknown for years. Without accompany- ing biological data, the discovery of new medicinal plant constituents is nothing more than pure phyto- chemistry. Yet, the search for specific pharmacolo- gic activities, e.g., the former plant antitumor pro- gram of the National Cancer Institute [1], often em- ploys bioassays which cost more money than is avai- lable for plant procurement and fractionation; in ad- dition, the search for specific activities often over- looks other useful activities which are not detected, or are ignored, in the screening process. There is a real need for reliable, general bioassays which can detect a broad spectrum of pharmacologic activities in higher plants and, yet, can be employed by natural Universidad Contral de Venezuela, Caracas, Venezuela * Visiting Assistant Professor from the Facultad de Farmacia, product chemists, in-house at low cost, to guide phy- tochemical screening and fractionation. Since most active plant principles are toxic at ele- vated doses, a possible approach to developing an ef- fective general bioassay might be simply to screen for substances that are toxic to zoologic systems, Once such substances have been isolated, a battery of spe- ific and more sophisticated bioassays could then be employed. Desiring a rapid, inexpensive, in-house, bioassay for screening and fractionation monitoring of our physiologically active plant extracts, we have been using a tiny crustacean, brine shrimp, as the ge- neral bioassay tool. The eggs of brine shrimp, Artemia salina Leacn, are readily available at low cost in pet shops as a food for tropical fish, and they remain viable for years in the dry state. Upon being placed in a brine solution, the eggs hatch within 48 hours, providing large num- bers of larvae (nauplii). Brine shrimp have been pre- viously utilized in various bioassay systems. Among these applications have been the analysis of pesticide residues [2-5], mycotoxins [6-11], stream pollu- tants [12], anesthetics [13], dinoflagellate toxins [14], morphine-like compounds [15], toxicity of oil disper- sants [16], cocarcinogenicity of phorbol esters [17], and toxicants in marine environments [18]. Most workers have made use of the hatched nauplii, alt- hough inhibition of hatching of the eggs has also been studied [19] In monitoring fractionation studies, brine shrimp have previously shown utility with fungal extracts di- rected at isolation of mycotoxins [20]. But for scree- ning and fractionation of active materials from hig- her plants, they have not been used. The organism is now suggested as a convenient probe for pharmaco- logic activities in plant extracts which may be manife- sted as toxicity towards the newly hatched nauplii Results and Discussion We have developed a method whereby com- pounds and extracts are tested at concentrations of,82 Meyer ot al Table | ‘We suggest that this simple bioassay system might Brine Shrimp Bioassay Results forKnown Active NaturalProducts be readily utilized by pharmacognosists and natural product chemists in the detection and isolation of natural compound LOsinugint higher plant constituents with a variety of pharmaco- logic activities. It has the advantages of being rapid podophy/toxin 24 (24 hours following introduction of shrimp), inex- eee aa a pensive, and simple (e.g., no aseptic techniques are — acy required). It easily utilizes a large number of conti- arene ties a nuously available organisms for statistical considera- Scie vesetea z8 tions and requires no special equipment or training stophenthin 218 and a relatively small amount of test sample (50 mg arbutin 215 for crude extracts). Active compounds thus obtained cattone 306, could then be subjected to more elaborate bioassays thymat 514 for specific pharmacologic activities. atropine SO, 686 santonin > 1000 10, 100, and 1000 jg/ml after being applied to filter paper discs which are placed in vials containing brine and ten shrimp in each of five replicates. Survivors are counted after 24 hours and the percentage of de~ aths at each dose is recorded. These data can then be used to estimate L's for comparison of potencies. To explore the range of natural products which could be detected in the bioassay, a number of known active natural compounds were tested. These results (Table 1) demonstrated the general utility of the bioassay with compounds of diverse structures and modes of activity. These results encouraged furt- her testing of the assay for its use in screening and eventual fractionation of active plant extracts. ‘As a demonstration project, the ethanol extracts of seeds of 41 species of Euphorbiaceae were tested; this plant family is well-known to contain toxic com- pounds of diverse structures [21]. Data for the bioas- say of these extracts are shown in Table IT; 9KB and 9PS cytotoxicities were determined and are shown for comparison [22, 23]. Eighteen of the cuphorb extracts displayed to ty (LCi < 1000 ug/ml) in the brine shrimp bioassay. Several of these are known to contain physiologically active principles (see Table II for references). Of 24 showed 9PS activity (LC) = 30 ug/ml), to brine shrimp. The brine shrimp bio assay is not specific for antitumor or, indeed, any particular physiological action; but for a significant number (14 of 24) of the species with cytotoxic activi- ty, it may be possible to monitor fractionation using principally the brine shrimp bioassay rather the more expensive and time-consuming cytotoxic assays. Oc- casional cross-monitoring in the more specific assay systems would be necessary to ensure that the activi- ties continue to correlate. The brine shrimp bioassay is clearly a more general test than the 9KB assay in which only six species were active (LCs) = 30 ug/ml) (Table II); only two of these showed significant acti- vity in the brine shrimp assay. Experimental Plant Materials ‘Samples of authenticated seeds of 41 species of Euphorbiaceae were obtained from the seed collection of the Northern Regional Research Laboratories of the United States Department of Agri- calture in Peoria, Mlinois, U.S.A. All samples were weighed, ground and defatted by shaking several times in hexane. The seeds ‘Wore then shaken several additional times with ethanol, and the ethanol extracts were concentrated in vacuo and weighed. ‘All pure compounds employed were obtained from commer- cial suppliers Optotoxicty Testing ‘9KB and 9PS cytotoxiciies were determined inthe Purdue Cell Culture Laboratory following protocols established by the Natio- nal Cancer Institute [23]. Activities for extracts are considered s- {nificant when LCs are = 30 gi Sample Preparation ‘Samples were prepared by dissolving 0 mg of compound or ex teact in Sm of methanol (Solution A) Solution B was prepared by AiatingO-S ml of A to 10 ma with methanol. Appropriate amounts ofsolution (10048, S0,IA, and S004 for 10, 100, and 1000 a! tl respectively) were transferred to 1.25 om (1/2in) discs of filter paper (Seueicnex and Scwuctt, no, 7404E). The discs were dred Iai, placed in 2 dram vials, ad then died farther in vacuo for One hour. Control diss were prepared using only methanol. Five replicates were prepared foreach dose level Hatching he Shrimp ‘Brine shrimp eggs (Living World, Metaframe Ine., Elmwood ark, N. J.07407, U.S.A.) were hatched in a shallow rectangular dish (22 « 32cm) filled with artificial sea water which was prepa- ‘red with a commercial silt mixture (Instant Oceans, Aquarium Sy- stem, Ine.) and double-disilled water. A plastic divider with se- veral/2.mm holes was clamped in the dish to make two unequal compartments, The eps (ca 50 mg) were sprinkled into the larger ‘compartment which was darkened, while the smaller compart- ‘ment was illuminated, After 48 hours the phototropic nauplii were collected by pipette from the lighted side, having been separated by the divider from ther shells Bioassay “Ten shrimp were transferred to each sample vial using» 9in di sposuble pipette (Scientific Products, diSPo pipettes), and art ‘ial sea water was added to make 5 ml. The naupliican be counted ‘macroscopically in the stem of the pipette against alighted back- ‘ground, A drop of dry yeast suspension (Red Star) (3 mg in S ml frtficial sea water) was added as food to each vial. The vials were ‘maintained under illumination, Survivors were counted, withtheBrine Shrimp Bioassay Table It Brine shrimp bioassay results of ethanolic extracts of soeds of Euphorbiaceae Percentdeaths at 24hr (05% Refer. Species 10 100° 1000 LCs, confidence 9PSLCs) 9KBLC;, encesto gil ygiml ug/ml ugiml interval’ girl gil Com ponents 1. Eremocarpus seigerus (Hoox) Bex. 92 78 100-24 (14-97)1.76x10% inact 2. Euphorbia amygdaloides Oe 007d 0-07) 68 inact, — 3. Croton tigi. 2 7898 = 8017-47), 1000 = 558 inact. 20. Euphorbia paralias 0 0 48 > 1000 7 20.2 inact 21. Euphorbia eriophora Boss. 0 4 48 > 1000 - 203 inact. 22. Trowia nudiforaL. 0 2 56 >1000 «= -- = agoxt0* 10% 23. Euphorbia heterophylla. 10-28 © 42> 1000 inact. 24. JatrophacurcasL 0 232 > 1000 9.0 25. Chidoscolus tepiquensis(Cosr.and 1a 23 = 40 > 1000 7A Ga.) Linoa 28. Daphniphylum humile Maso. Oeeees0) 4 > 1000 inact, act 27. Mallotusphilipensis La.) Moca. -ARo. (OseeesOaeee toe 1000) inact inact 28. Chrozophora tnctoria (L.)A. Juss. Drees) 4 >1000 inact. inact 29. Sapium haematospermum Musu.-ARc. (Obes sO 1088 1000] 62.4 act 30. Jatropha gossypifoliaL. a) 0 > 1000 inact, inact 31. EuphorbiafatcataL. 0 0 ~~ 6 1000 inact. inact 82. Excoecaria bussel (Pax) Pax 0 0 16 > 1000 129 inact. 33. Bischotlajavanica Bune ar) 2 >1000 389 inact ‘34. Macaranga perakensis Hook 0 19 © 10 > 1000 78 inact. 35. Manihotisoloba Srave.sy a) 6 > 1000 4033 36. Cnidoscoluselastcus Lunoe. a) 0 > 1000 335312 37. Manihot woodieana Mua 8 4 B10 82 414 38. Sapium sebiferum(L.) Ross. 0 14 ©. >1000 082 inact 39. Aleurites moluccane(L.) Wixo. 4 10 © 16 > 1000 aye on 40. Euphorbiamedicaginea Bais, fpeeees0) 0 > 1000 40, 599 41. Euphorbiamyrsinies . Oty ar inact, inact * Where data were insuficient or probit analysis, LCs», were estimated using logit iransformation (s9@ text) which does not provide con fidence interval. sid of a3 x magnifying glass, after 6 and 24hours, and the percent ‘deaths at each dose and control were determined, The 24 hours ‘counts were more useful. In cases where control deaths occurred, the data were corrected using Abbotts formula [32] % deaths = [(test—controlicontrol] 100. Cp Determinations Eca's and 95 % confidence intervals were determined from ‘the 24 hour counts using the probit analysis method described by Fivwey [33]. In cases where data were insufficient for ths techni- que, the dose-response data were transformed into a straight line by meansof a logit transformation [34]; the LD, was derived from the best fit line obtained by linear regression analysis34 Meyer et al Acknowledgements [BNM acknowledges support as the AFPE Charles J. Lynn Me- ‘moral Fellow. Thanks are expressed toC. R. Surm.Jr.,and R. G. Powe, USDA, Peoria, for providing the seeds. This work was supported in part, by BMRG no, SOT-RR-OSS86 from the Natio= nal Institutes of Health. NRF acknowledges a Research Scholars- hip from Consejo Desarrollo Cientifico y Humanistico, Universi= dad Central de Venezuela. References (2) Sutinss, Mand J. 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