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Neriifolin and 2' -Acetylneriifolin: Insecticidal and Cytotoxic Agents of 1 Thevetia thevetioides Seeds ,2

J. L. McLAUGHLIN, B. FREEDMAN, R. G. POWELL, AND C. R. SMITH, JR. Northern Regional Research Center, Agric. Res., SEA, USDA, Peoria, IL 61604

Reprillted from the JOURNAL OF ECONOMIC ENTOMOLOGY Neriifolin and 2' -Acetylneriifolin: Insecticidal and Cytotoxic Agents of 1 Thevetia thevetioides Seeds ,2

J. L. McLAUGHLIN, B. FREEDMAN, R. G. POWELL, AND C. R. SMITH, JR. Northern Regional Research Center, Agric. Res., SEA, USDA, Peoria, IL 61604 ABSTRACT J. Ecan. Enlama!. 73: 398-402 (1980) A bioassay procedure utilizing the European com borer, Ostrinia nubilalis (Hiibner), has been used to guide the phytochemical fractionation of active extracts of the seeds of a yellow oleander, Thevetia thevetioides (HBK.) K. Schum. (Apocynaceae). The known cardiotonic glycosides, neriifolin and 2'-acetylneriifolin, were crystallized as the active insecticidal agents, giving LDso determinations of 30 ppm and 192 ppm, respectively, when incorporated into the com borer diet. These compounds also exhibited cytotoxic activities of 2.2x 10-2 and 3.3 x 10-2 fLg/ml, respectively, in the KB (human nasopharynx epidermoid carcinoma) in vitro system. The glucoside of f3-sitosterol also was isolated but it lacked insecticidal activity.

Freedman et al. (1979) reported that ethanol extracts Materials and Methods of the seeds of a Mexican yellow oleander, Thevetia Plant Material thevetioides (HBK.) K. Schum. (Apocynaceae) were lethal (100% larval mortality) when incorporated into Seeds of T. thevetioides were collected in September the diet of the European com borer, Ostrinia nubilalis 1977 in Mexico for the USDA Medicinal Plant Re­ (Hiibner). sources Laboratory through which voucher specimens Toxicity of Thevetia species to higher animals, in­ (B-171053, PR-48977, colI. no. ES-106) are preserved. cluding humans, is well documented (Kingsbury 1964) The seeds were pulverized in a Wiley mill. and is attributed to cardiotonic glycosides (Zechner European Com Borer Bioassay 1966) whose steroidal aglycone is digitoxigenin (Cruz Com borer larvae were reared from eggs supplied by et al. 1977). In many tropical areas of the world, where the Com Insects Res. Unit, USDA, Ankeny, lA. Bioas­ the plants are both endemic and naturalized, Thevetia says were performed, essentially as described by Freed­ species have several folkloric medicinal uses (Watt and man et al. (1979), using plastic jelly cups each contain­ Breyer-Brandwijk 1962), including the treatment of tu­ ing five 7-day-old larvae and 4.0±0.1 g of diet mixed mors (Hartwell 1967, Martinez 1969). with the appropriate dose of treatment material. Crude In India, extracts of Thevetia leaves are used as a extracts were tested at 40 mg/cup, the 1st column frac­ pediculicide (Atal and Kapur 1977); in Mexico, the tions at 5 mg/cup, subsequent column fractions at 2 mg/ milky juice is applied to the mange (itch mites) (Mar­ cup, mother liquors at 1 mg/cup, and crystalline isolates tinez 1969); and powdered seeds of Thevetia act as a at a range of 2-0.03125 mg/cup. protectant of stored seeds against insect infestations Each treatment was replicated 4 times. Mortalities (Pandey et al. 1977). Several papers report effectiveness were determined at 5 and 9 days by counting survivors. as well as ineffectiveness of Thevetia powders and ex­ Activities are expressed as percent mortality at 9 days tracts against a variety of insect species (McIndoo 1945, adjusted for control mortality by Abbott's formula; con­ Jacobson 1958, 1975, Deshmukh and Borle 1975). trols were run concurrently for the various solvents Without reporting experimental data, Gattefosse (1949) (acetone, ethanol, or petroleum ether) used to apply the attributed t'le insecticidal activity of T. neriifolia Juss. treatments to the diet. Estimations of LDso values were (synonym: T. peruviana (Pers.) Schum.) to the glyco­ made using the standard probit analysis ofDaum (1970). side, thevetin, and to another unidentified material of even greater toxicity; he also proposed that these ma­ Cytotoxicity Bioassay terials be developed as a new agricultural insecticide. Activity of preliminary extracts and crystalline com­ Our purpose was to isolate and characterize the in­ pounds against KB cells in vitro was determined by secticidal compound(s) of Thevetia. The com borer contractors for the National Cancer Institute employing bioassay was used as a guide to the phytochemical frac­ standard protocols (Geran et al. 1972). Isolated com­ tionation of the active ethanol extracts of the seeds of pounds are considered active if the EDso is ~4 fLg/mI. T. thevetioides. Concurrently, certain extracts and frac­ Chromatographic Methods tions also were found to exhibit significant cytotoxic Column chromatography was performed in appropri­ activity against KB (human nasopharynx epidermoid ately sized columns using silica gel (Hi-Flosil, 60/200 carcinoma) cells (Geran et aI. 1972), and we wished to mesh, Applied Science Laboratories, State College, PA) determine if the final insecticidal isolates also might be packed in chloroform slurries. Column fractions were the active cytotoxic agents. assayed with TLC on silica gel (silica gel 60 F254 , 0.25­ mm precoated plates, EM Laboratories, Elmsford, NY). The TLC solvent system was chloroform-methanol (9:1), ~on of firm names or trade products does not imply endorsement or and the developed chromatograms were visualized with recommendation by the USDA over other fIrms or similar products not mentioned. , Received for publication Jan. 14. 1980. chromic acid-sulfuric acid char (Stahl 1969). 398 June 1980 McLAUGHLIN ET AL.: INSECTICIDAL AND CYTOTOXIC AGENTS OF Thevetia 399

Characterization Methods and 100% methanol in chloroform. A total of ninety 25­ Melting points were determined with a Fisher-Johns ml fractions was collected and combined after TLC. melting point apparatus and are uncorrected. Infrared Significant activities (at 2 mg/cup) were detected in frac­ spectra were recorded in chloroform on a Perkin-Elmer tion 2:20-26 (2.28 g, 54.5%) and fraction 2:27-32 (0.32 model 700 IR spectrometer. 90 MHz IH and proton de­ g,56.7%). coupled 13C nrm spectra were obtained in deuterochlo­ Fraction 2:20-26 (2.27 g) was then chromatographed roform using a Brunker WH-90 instrument with tetra­ on a 2.0x60-cm column of silica gel (42.9 g) developed methylsilane as the internal reference. Authentic samples with 250-ml portions of 0, 0.5, 1.0, 1.5, 2.0, 2.5, 5, of neriifolin and 2'acetylneriifolin were supplied by Dr. 25, and 100% methanol in chloroform. A total of ninety­ Jose Iriarte, Research Division, Syntex SA, Mexico 10, one 25-ml fractions was collected and combined after D.F. Mexico. TLC. The most significant larvicidal activity (at 2 mg/ cup) was found in fraction 3:36-46 (1.50 g, 72.9%). Isolation Procedures and Results Fraction 3:36-46 (1.49 g) was chromatographed on Extraction ofPlant Material a 2.0x60-cm column of silica gel (42.7 g) developed A total of 18.75 kg of the powdered plant material successively with 250-ml portions of chloroform-meth­ was extracted for 6 h with hexane in a large pilot plant anol mixtures containing 0,0.25,0.5,0.75, 1.0, 1.5, Soxhlet extractor, and the inactive hexane extract was 2, 5, and 100% methanol. A total of ninety-one 25-ml discarded. The dried marc was then extracted (Soxhlet) fractions was again collected and combined after TLC for 6 h with ethanol, and the ethanol extract was con­ analysis. The most significant activity (at 2 mg/cup) was densed to a syrup (1.09 kg) under vacuum in a rotary found in fraction 4:57-64 (1.24 g, 87.3%). evaporator. A portion (435.2 g) of the ethanol extract Fraction 4:57-64 crystallized upon standing and was (corn borer activity, 94.0%) was treated with 8 liter of recrystallized from ethyl ether, yielding 0.40 g, mp water and was partitioned 3 times with 8-liter portions 211°-214°C. After further recrystallization, 0.38 g was of chloroform. The freeze-dried residue (204.0 g) from obtained, mp 220°-222°C. This compound was active the aqueous layer was inactive, whereas the syrup re­ with an LD50 of 0.77 mg/cup (192 ppm in the diet, slope maining after vacuum evaporation of the combined chlo­ 0.83) with 95% limits from 0040 to 2.17 mg/cup. The roform layers was very active (100%). activity of the mother liquor (36.5% at 1 mg/cup) was The entire chloroform residue (199.6 g) was parti­ entirely due to the presence of this compound and in­ tioned between hexane and methanol-water (9: I) (750 dicated the absence of more potent compounds in this ml each); each layer was backwashed twice with two fraction. This active crystalline isolate was subsequently 250-ml portions of the respective immiscible solvents. identified as 2'-acetylneriifolin (Fig. 1). The hexane residue (155.9 g) was inactive, whereas the isolation ofNeriijolin from Fractions I :31-34 and methanol-water residue was highly active (100% at 40 1:35-39 mg/cup). Fraction 1:31-34 (8.20 g) was chromatographed on Chromatography ofActive Crude Extract a 4.5x67-cm column of silica gel (205 g) developed The entire methanol-water residue (49.2 g) was chro­ with 500-ml portions of 0,2.5, 5, 7.5, 10, 12.5, 15, matographed on a 6x72-cm column containing 467 g 25, 50, and 100% methanol in chloroform. A total of ofsilica gel. Development was made with one-liter por­ 100 fifty-ml fractions was collected and combined ac­ tions of solvent mixtures containing 0, 5, 10, 15, 25, cording to TLC analyses. Activity (2 mg/cup) was de­ and 50% methanol in chloroform followed by rinses tected in fractions 5:38-39 (3.76 g, 94.1%) and 5:40-43 with 3 liters of methanol and one liter of methanol-water (2048 g, 94.3%); the active substance crystallized upon (9:1). A total of 90 fractions of 100 ml each was col­ standing. Additional significant activity was detected in lected followed by the rinse fractions of 750 ml, one fraction 5:44-49 (1.11 g, 82.0%), but TLC indicated liter, and 600 ml. Fractions were combined on the basis that this was due to the same active compound. of similarity upon TLC analysis. The crystalline residues of fractions 5:38-39 and 5:40­ Significant activity (at 5 mg/cup) was found in 3 of 43 were recrystallized twice from boiling chloroform the combined column fractions designated as fraction (0.41 and 0.23 g, respectively; mp 214°-218°C), and 1:29-30 (3.03 g, 100%), fraction 1:31-34 (8.27 g, 2nd crops of less pure (mp and TLC) crystals were ob­ 100%), and fraction 1:35-39 (7042 g, 79.6%). Each of tained from the mother liquors. This compound was ac­

these active fractions was subsequently resolved by ad­ tive with an LD50 of 0.12 mg/cup (30 ppm in the diet, ditional column chromatography. An inactive amor­ slope 1.65) with 95% limits from 0.01 to 0.29 mg/cup. phous powder, which formed upon condensing fraction Bioassays of the mother liquors (both at 62.2% at 1 mg/ 1:40-44, was insoluble in all common solvents with the cup) again indicated that this crystalline compound was exception of pyridine; this material was identified eH the only active agent in these fractions. This compound nmr and 13C nrm) as ,B-sitosterol glucoside [(3,B)-stig­ was subsequently identified as neriifolin (Fig. 1). mast-5-en-3-yl-,B-D-glucopyranoside] [mp 304° (de­ Additional neriifolin (0.29 g), obtained by 2 similar comp.); lit. mp 305°C (decomp.) Karrer 1958]. column chromatographic separations, accounted for the larvicidal activity observed in fraction 1:35-39. Isolation of2'-Acetylneriijolin from Fraction 1:29-30 Fraction 1:29-30 (2.89 g) was chromatographed on Identification ofNeriijolin a 2.6x47-cm column of silica gel (43.5 g) developed The active crystalline compound from fractions 1:31­ with 250-ml portions of 0,2.5,5,7.5, 10, 15,25,50, 34 and 1:35-39 gave a mp (214°-218°C) similar to that 400 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 73, no. 3 o """-'--0 23 21

16 HO 15 14 OH o 4 6 FIG. I.-Structures of neriifolin (R '" H) and 2'-acetylneriifolin (R '" COCH3).

reported for neriifolin (lit. mp 204°-214°C, Cruz et al. erence 2 '-acetyineriifolin cochromatographed (Rr 0.48) 1977; mp 218°-225°C, Zechner 1966). The lH nmr on TLC and gave identical visualization chromophores. spectrum was essentially identical to the spectrum of a reference sample of neriifolin: a 5.88 (t, 1, H-22, J = Discussion 1.7 Hz), a4.87 (s, I, H-I '), a4.92 (ddd, 2, H-21,21', The European corn borer bioassay successfully guided J = 18.8, 1.7 Hz), a3.9 (bm, I, H-3), a3.69 (s, 3, ­ the phytochemical fractionation of the active extracts of OCH3), a 2.78 (bm, 1, H-17), a 1.26 (d, 3, H-6', J = the seeds of T. thevetioides to yield 2 active insecticidal 6.0 Hz), a 0.97 (s, 3, H-19), a 0.88 (s, 3, H-I8). 13C compounds. Identifications of the active compounds as nmr peaks were tentatively assigned (Table 1), and the neriifolin and 2 '-acetylneriifolin (synonyms: cerberin, IR spectrum gave the expected peaks: 3610 cm-l (broad, l veneiferin) (Fig. 1) were made by using physical and hydroxyl), 1790 and 1750 cm- (doublet, lower band spectral data. A doublet in the carbonyl region of the IR stronger, butenolide carbonyl). The isolated and refer­ was, with each compound, distinctive for the a,f3-un­ ence neriifolin cochromatographed (Rr 0.35) on TLC saturated y-lactone moiety (Nakanishi 1962). Although and gave identical chromophores when visualized with the previously reported lH nmr spectrum of neriifolin the spray reagent. was determined in deuterated methanol and was not di­ Identification of2'-Acetylneriifolin rectly comparable to our spectra in deuterochloroform, The crystalline active compound from fraction 1:29­ the spectrum of 2'-acetylneriifolin was quite comparable 30 gave a mp (220°-222°C) similar to that reported for to the literature values (Cruz et al. 1977). l3C nmr spec­ 2'-acetylneriifolin (mp 218°-220°C, Cruz et al. 1977; tra for these glycosides have not been previously pub­ mp 212°-215°C, Zechner 1966). The lH nmr spectrum lished, and tentative assignments are listed in Table 1; was essentially indistinguishable from that of reference l3C nmr spectra of 3-acetyldigitoxigenin and several al­ 2 '-acetylneriifolin:a 5.87 (t, I, H-22, J = 1.8 Hz), a doses served as models (Wehrli and Nishida 1979, Gorin 5.06 (d, I, H-I', J = 3.9 Hz), a4.89 (apparent q3, 2, and Mazurek 1975, Tori et al. 1973); the shift for the H-2I ,21', J = 1.8 Hz), a 4.64 (q, 1, H-2', J = 3.9, 3'-O-methyl carbon was comparable to values reported 9.6 Hz), a 3.88 (bm, I, H-3), a 2.78 (bm, I, H-17), for this group on other sugars (Milikovic et al. 1974); a2.07 (s, 3-COCH3), a 1.26 (d, 3, H-6', J = 6.4), a and the methyl at 6' on the thevetose was easily ob­ 0.96 (s, 3, H-19), 00.88 (s, 3, H-18). Table 1 lists the served in each glycoside as the most upfield signal. A l3C nmr assignments. The IR spectrum was quite similar final direct comparison of the isolated compounds with to that of the isolated neriifolin: 3610 cm-l (broad, hy­ reference compounds by melting point and TLC con­ droxyl), 1785 and 1745 cm-l (doublet, lower band firmed the identifications. A 3rd isolated but inactive stronger, butenolide plus ester). The isolated and ref- compound was identified by lH nmr, l3C nmr, and mp as f3-sitosterol glucoside, a common substance in higher ~B quanets evident with neriifolin are obscured here. plants (Karrer 1958). June 1980 McLAUGHLIN ET AL.: INSECTICIDAL AND CYTOTOXIC AGENTS OF Thevetia 401

Table I._13C nmr assignments for neriifolin and 2'-acetylneriifolin using 3-acetyldigitoxigenin as a modela •

Carbon no. Neriifolin 2'-Acetylneriifolin 3-Acetyldigitoxigenin

1 30.09 29.70 30.8 2 26.65 26.71 25.4 3 71.75 73.57 71.4 4 30.74 30.42 30.8 5 36.98 36.52 37.4 6 26.65 26.71 26.8 7 21.45 21.38 21.6 8 41.98 41.92 41.8 9 35.87 35.74 36.1 10 35.29 35.22 35.8 11 21.25 21.38 21.6 12 40.10 40.10 40.3 13 49.65 49.72 50.3 14 85.59 85.59 85.6 15 33.28 33.21 33.0 16 26.97 26.97 27.3 17 51.02 51.02 51.5 18 17.55 17.61 16.0 19 23.92 23.98 23.9 20 174.37 174.76 177.1 21 74.87 74.41 74.7 22 117.83 117.70 117.4 23 174.37 174.76 176.3 I' 97.36 93.85 2' 73.44 75.39 3' 84.25 81.04 4' 73.44 72.33 5' 67.65 67.13 6' 15.79 15.86 OCH3 60.51 60.57 CO on Ac 170.34 CH3 on Ac 20.99

, Tori el aJ. (1973). Results of the corn borer bioassay correlated closely respectively, Zechner 1966), and inhibition of Na+­ with the concentrations of neriifolin or 2'-acetylneri­ K+ATPase in cell membranes affecting cation transport ifolin in the various fractions. Thevetin (neriifolin-4 '­ is most likely the molecular basis for their mode of gentiobioside), reported as insecticidal by Gattefosse action (Goth 1972). Their mode of action in insects is (1949), was not detected; neriifolin, the more active and unknown but may involve similar effects on ion trans­ abundant of the 2 active isolates, is likely the more port in the susceptible species. Some indication of an­ toxic, unidentified, insecticide reported by Gattefosse tifeedant activity at lower doses was noted in the bioas­ (1949). Neriifolin (LD50 0.12 mg/cup, 30 ppm in the say because any living com borer larvae were very diet) was ca. 6 times as active as 2 '-acetylneriifolin small; however, these observations were not quantitated. (LD50 0.77 mg/cup, 192 ppm in the diet) and showed The 2 insecticidal glycosides also were significantly an excellent dose:response relationship with larval mor­ active in the KB in vitro cytotoxicity assay (neriifolin 2 tality. Under similar conditions carbofuran, a popular ED50 2.2X 10- JLg/ml and 2'-acetylneriifolin ED50 2 commercial insecticide for corn borers, has an LD 50 of 3.3X 10- JLg/ml),and their presence explains the cyto­ 2 ca. 1-2 ppm. toxicities (ED50 as low as 2.0x 10- JLg/ml) observed In their comprehenisve summaries of insecticidal ac­ with preliminary crude extracts. Neriifolin, without tivities found in plants, McIndoon(1945) and Jacobson quantitative data, has been reported as being active in (1958, 1975) reported that various extracts of Thevetia vivo against Lewis lung carcinoma (LL) and sarcoma species are very effective against certain insects (Amer­ 180 (SA), and in vitro against KB; it is inactive in vivo ican cockroaches, black carpet beetle larvae, adult chry­ against L-121O lymphoid leukemia (LE), P388 lympho­ santhemum aphids, potato aphids, and codling moth lar­ cytic leukemia (PS), and Walker carcinosarcoma (WA); vae) but only somewhat effective or completely 2'-acetylneriifolin (cerberin) has reported KB and weak ineffective against others (German cockroaches, milk­ (130% TIC) PS activity (Hartwell 1976). Our prelimi­ weed bugs, confused flour beetles, webbing clothes nary KB active crude extracts, which contained the gly~ moth larvae, Aedes mosquito larvae and adults, saw­ cosides, were inactive in vivo against colon 38 (C8), tooth grain beetles, Mexican bean beetle larvae, hou­ B16 melanocarcinoma (Bl), and PS. The cytotoxicities seflies, and caterpillars). Thus, it can be predicted that of many cardenolides have been previously noted (Har­ the insecticidal activities of neriifolin and 2 '-acetylner­ twell 1976), and these compounds continue to be iso­ iifolin may be limited to only certain insect species. In lated as potential antitumor agents (Hembree et al. mammals, the glycosides are cardiotoxic and are quite 1979). potent (LD50 in cats, O. 196 mg/kg and 0.147 mg/kg, The folkloric uses of Thevetia plant materials as in- 402 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 73, no. 3 secticides and antitumor agents are thus explainable by tra of aldoses and derived methyl glycosides. Can. J. the presence of the 2 isolated cardenolides, neriifolin Chern. 53: 1212-23. and 2'-acetylneriifolin, as the active principles. Fur­ Goth, A. 1972. Medical Pharmacology. 6th ed. C. V. Mosby thermore, the potent insecticidal activity of neriifolin Co., St. Louis, MO. 704 pp. . Hartwell, J. L. 1967. Plants used against cancer. A survey. against the European com borer would justify studies Lloydia 30: 379-436. with additional insect species and exploration of its po­ 1976. Types of anticancer agents isolated from plants. Can­ tential for development. cer Treatment Rep. 60: 1031-67. Hembree, J. A., C.-j. Chang, J. L. McLaughlin, G. Peck, and J. M. Cassady. 1979. Potential antitumor agents: a cytotoxic cardenolide from Coronilla varia. J. Nat. Prod. Acknowledgment 42: 293-8. We acknowledge the technical assistance of Milton Jacobson, M. 1958. Insecticides from plants, a review of the J. Axley and Mark P. Detweiler and thank Richard V. literature, 1941-1953. USDA Agric. Handb. 154: 299pp. Madrigal and William F. Kwolek for assistance with the 1975. Insecticides from plants, a review of the literature, 1954-1971. Ibid. 461: 138 pp. com borer bioassay. Dr. Robert E. Perdue, USDA, Karrer, W. 1958. Konstitution and Vorkommen der organ­ Beltsville, MD, provided the T. thevetioides seeds. Par­ ischen Pflanzenstoffe. Birkhauser Verlag, Stuttgart. 1207 tial support to J. L. McLaughlin was provided by con­ pp. tract no. NOI-CM-97296, Division of Cancer Treat­ Kingsbury, J. M. 1%4. Poisonous plants of the United States ment, National Cancer Institute. and Canada. Prentice-Hall, Englewood Cliffs, NJ. 626 pp. Martinez, M. 1%9. Las Plantas Medicinales de Mexico. Quinta edicion. Ediciones Botas, Mexico, D.F. 656 pp. Mclndoo, N. E. 1945. Plants of possible insecticidal value, REFERENCES CITED a review of the literature up to 1941. USDA, Bureau En­ tomol. Plant Quarantine E-661: 286 pp. Atal, C. K., and B. N. Kapur. 1977. Cultivation and Utili­ Milikovic, M., M. 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