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 of Medicinal Chamistry and Pharmacognosy, Schoo! of Pharmacy and Pharmacal Sciences, and Coll Culture Laboratory, Purdue Cancer Center, Purdue University, West Lafayette, IN 47807 ceived: January 10, 1962 Key Word Indes Artemia salina; Brine shrimp; Bioassay methods; Euphorbiaceae seeds; 9KB and 9PS cytotoxicities. Abstract A method, utilizing brine shrimp (Artemia salina Leacx), is proposed as a simple bioassay for natural product research. The procedure determines LCsq 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~ Hiable, 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 * Visiting Assistant Professor from the Facultad de Farmacia, Universidad Central de Venezuela, Caracas, Venezuela. 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- cific and more sophisticated bioassays could then be employed. Desiring a rapid, inexpensive, in-house, Dioassay 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 Leacu, 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 eggshasalso 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a2 Meyer et al Table | ‘We suggest that this simple bioassay system might Brine Shrimp Bloassay Results for Known Active NaturalProducts be readily utilized by pharmacognosists and natural roduct chemists in the detection and isolation of natural compound LCssin natn + plant constituents with a variety of pharmaco- logic activities. It has the advantages of being rapid podophyotoxin 24 (24 hours following introduction of shrimp), inex- Derberinachlowida 228 ensive, and simple (¢.g., no aseptic techniques are styehninesultate 72 Pent ple (e'g.. no asept ques are i a required). It easily utilizes a large number of conti- ieemice ae nuously available organisms for statistical considera- Ghecincutcte ae tions and requires no special equipment or training stophanthin 216 and a relatively small amount of test sample (50 mg arutin 275 for crude extracts). Active compounds thus obtained cating 308 could then be subjected to more elaborate bioassays thymo! sia for specific pharmacologic activities. atropine SO, 638, santorin >1000 10, 100, and 1000 g/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 LCs 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 sereening and eventual fractionation of active plant extracts ‘As a demonstration project, the ethanol extracts of sceds 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 euphorb extracts displayed toxici- ty (LCyq < 1000 g/ml) in the brine shrimp bioassay. Several of these are known to contain physiologically active principles (see Table II for references). Of 24 species which showed 9PS activity (LCz1= 30 ug/ml), 14 were toxic 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 (LC3y = 30 pg/ml) (Table T1); only two of these showed significant acti- vity in the brine shrimp assay. Experimental Plant Materials ‘Sumples of authenticated seeds of 1 species of Euphorbiaceae were obtained from the seed collection ofthe Northern Regional Research Laboratories ofthe United States Department of Agri- culture in Peoria, Ilinois, 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 eommer- cial suppliers. Cytoroxicity Testing ‘KB and 9PS cytotoxiciies were determinedin the Purdue Cell Culture Laboratory following protocols established by the Natio- nal Cancer Institute [23}, Activities for extracts are considered s ‘nificant when LCy's are = 30 ug/ml. Sample Preparation ‘Samples were prepared by dissolving $0 mg ofcompound or ex- ‘ractin ml af methanol (Solution A), Solution B was prepared by diluting 0.5 ml of Ato 10m with methanol. Appropriate amounts ‘of solution (100 1B, 30 A, and'SO0 LA for 10, 100,and 1000 wg! ‘ml, respectively) were tansierred to 1.25.m (12 in) discs of filter paper (Semone and Scworts, no. 740-E). The discs were dried Imai, placed in 2 dram vials, and then dried further in vacuo for ‘one hour. Control discs were prepared using only methanol. Five replicates were prepared foreach dose level Hatching the Shrimp ‘Brine shrimp eggs (Living World, Metaframe Ine., Elmwood Park, N. J.07407, U.S.A.) were hatched ina shallow rectangular ish (22 » 32cm) filed with artificial sea water which was prepa- ‘red with «commercial salt mixture (Instant Oveans, Aquarium Sy- stom, Inc.) and double-istilled water. A plastic divider with se- veral!2 min holes was clamped in the dish to make two unequal ‘compartments. The eg (ca. 50 mg) were sprinkled into the larger ‘compartment which was darkened, while the smaller compart- ‘ment wasilluminated, After 4 hours the phototropic nauplii were collected by pipette from the lighted side, having been separated by the divider from their shells Bioassay "Ten sbrimp were transferred to each sample vial using a9 in di- sposable pipette (Scientific Products, diSPo pipettes), and artifi- ‘dal sea water was addled to make 5 mi The naupliican be counted ‘macroscopically in the stem of the pipette against alighted back- {ground, A drop of dry yeast suspension (Red Star) ( mgin Smal Artifical sea water) was added as food to each val. The vials were jined wader illumination. Survivors were counted, withtheBrine Shrimp Bioassay = Table Il Brine shrimp bioassay results of ethanolic extracts of seeds of Euphorbiaceae Percontdaaths at 24 hr (95% eter Species 10 100 1000 LOjp confidence SPSLOs) SKBLOs) encesto gin! pgm! gi! gil intervaly” = gin! git Com ponents 1, Eremocarpus setigerus Hoos) Box 32 78 100, 2k (14-87) 1.78x10* inact 2. Euphorbia amygdaloides 30 874 80 BB) 88 inact. 3. CrotontigiumL. 2% 78 807-47) < 10% =< 108 4. Rleverehoniaarenaria A. Grav 2 44 «= 1008 — inact. inact. 5. Seplummontevidense Kiorzsen 0 24 98116 (5-219) 4.2 inact. 6. Euphorbia lagascae Srreno. 30 4 «= 8118 (85-268) «0.8 nck 7, Euphorbia marginata Purs 0 «2% «= 100162 (120-227) «77408 8. Aleurites ford Heus.. 0 10 «100 «a7 - 183 inact 8. Antidesmanigricans Tu. 0 4 «100 286 - inact, inact. 10. Euphorbia lathyris. 0 6 «BBG (2B~465) 12 74D 41. Bridalaretusa(.) Soren, 6 14 100868 - 78 4A 12, Euphorbia cyparissias L. 0 24 = 72869 (24-594) 24x10? 3.8 48, Sapium japonicum Pax, and K, Horr. 9 10 8386. ((271~566) 31.9 inact 14. Euphorbia cybirensis Bos. 0 8 mM 516 63-754 6B HSA 18. Chrozophorahierosolymitana Serexs, 0 0 58 BY (456-947) 855 inact. 16. Patranjivaroxturghi Wa. 0 8 = 6713 (462=1290) inact. inact. 17. Daphniohyllum himalaenso (Ben) rn a) nt inact. inact, Mum. -Asa, 18. Jatropha spathulata Mucu.-Aec. 10 82 48981 (874-7763) 0.27 inact 18, Manihot rubricaulis |. M. lxneron 0 0 38 > 4000 - 558 inact, 20. Euphorbiaparatias 0 0 48 1000 ~ 202 inact 21. Euphorbia eriophora Boss. 0 4 = 48 1000 ~ 303 inact 22. Trowianudifloral.. 0 2 58 1000 —gg0x10* 10% 2, Euphorbia heterophylla. 10 28 © 42 > 1000 inact. 24. datrophacurcasl. 0 2 82 S100 30 25, Cnidoscolus epiquensis(Cosr.and 13-23 = 40 > 1000 7A Ga) Loa 28, Daphniphylum hurnte Muses, 0 0 4 1000 lnact. inact 27. Mallotusphilipensis Lou.) Mucu.-Ana. 0 6 12 > 1000 inact. inact 28, Chrozophora tnctoria(L.)A. Juss. 0 0 4 1000 inact. inact 29. Saplum haematospermum Mues.-Ars. 0 = ~~ 10 > 1000 524 inact 90. Jatropha gossypitola\. 9 9 0 >1000 inact. inact 31. Euphorbia falcata L. 0 0 6 >1000 inact. inact 82, Excoscaria bussel (Pax) Pax 9 0 16 >1000 129 inact 88. Bischotiajavanica Buse 0 0 ~~ 2 51000 389 inact, 34, Macaranga perakensis Hoox 0 10 © 10 > 1000 78 inact. 35. Maninotisoloba Sravoxv 0 0 6 >1000 4033 386. Cnidoscoluselasticus Line. 0 9 0 >s000 335312 37. Manihot tweodicana Mua. 8 4 S400 52 414 38. Saplumsebiferum(L.) Rom. 0 14 = > 1000 082 inact 89. Aleurites moluecana (L.) Wao. 4 10 © 16 >1000 53268 40. EuphorbiamedicagineaBoss, 9 0 9 >1000 40, 5a 41. Euphorbia myrsinites 0 0 =~» >1000 inact.” inact * Where data were insuficentfor probit analysis, LCzy, were estimated using log transformation (see text) which does nol provide con- fidence intervals. aid of a3 X magnifying gass,after6 and24 hours, and the percent ‘deaths at each dose and control were determined, ‘The 24 hours counts were more useful. Ia cases where control deaths occurred the data were corrected using Abbot's formula [32]: % deaths = [(Gest~controlfeontrol} x 100, LC q Determinations C's and 95 % confidence intervals were determined from the 24 hour counts using the probit analysis method described by Frwy [33]. In eases where data were insufficient for this techni- ‘que, the dose-response data were transformed into a straight line bby means of alogittransformation [34]; the LD. was derived from the best fit ine obtained by liner regression analysis