Toxic phorbol esters from Chinese tallow stimulate protein kinase C

Toxiona, Vol . 25, No. 11, pp. 1229-1233 . 1987. Printed in Great Britain .

W41-0101187 s3 .00+ .00 ® 1997 Perproon Journals Ltd.

TOXIC PHORBOL ESTERS FROM CHINESE TALLOW STIMULATE PROTEIN KINASE C G. BROOKS,' N. A. MORRICE,2 C. ELLIS,2 A. AITKEN,2 A. T. EVANS' and F. J. EVANS'*

'Department of Pharmacognosy and Pharmaceutical Chemistry, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WCIN IAX, U.K ., and 'Laboratory of Protein Structure, National Institute for Medical Research, Mill Hill, London NW7 IAA, U.K . (Accepted forpublication

19 May 1987)

G. BROOKS, N. A. MORRIcE, C. E,.las, A. ArncEN, A. T. EvAm and F. J. EVAm . Toxic phorbol esters from Chinese tallow stimulate protein kinase C. Taxiton 25, 1129 -1233, 1987 . - Phorbol esters were isolated from the seeds of Chinese tallow (Sapium seb(ferum L. Roxb .) . These compounds were based on the tigliane nuclei, 4-deoxyphorbol, 12-deoxyphorbol and 4,20dideoxy-5-hydroxyphorbol . The pro-inflammatory activity (m_a) of the pure compounds was between 0.042 and 2.6 nmoles per ear. Protein kinase C activation assays were carried out on samples of enzyme purified from mammalian brain and the activities (Kj were in the range 76-176 nM . The 4,20-dideoxy-S-hydroxy analogue was inactive in both tests. Chinese tallow, which is used as a substitute for linseed oil, may represent an industrial toxic hazard in terms of both pro-inflammatory and tumour-promoting effects . INTRODUCTION

are a large group of natural products which are well known for both their pro-inflammatory and their tumour-promoting activities in vivo (EVANS, 1986a) . Currently no government legislation exists for the control of tumour-promoting agents in commercially available products manufactured from plants containing these substances . Plants which are known to contain phorbol esters derive mainly from the large family Euphorbiaceae. The Chinese tallow tree (Sapium sebtferum) is a euphorbiaceous plant which is used in the manufacture of paints, varnish, cosmetics, fuel oil, timber and as a drug (RADCLIFFE-SMITH, 1986). Ingestion of the fruits has also been associated with a number of human poisonings (ELLIS, M. D., 1978). OHIGASHI et al., (1983) have demonstrated positive Epstein-Barr viral expression assays by extracts of this plant and have implicated Chinese tallow as a causative agent in human cancers, a conclusion supported by the detection of trace amounts of 12-deoxyphorbol in the seeds (SEiP et al., 1983) . Our initial unpublished investigations of the plant did not confirm these findings and consequently there was a possibility that the Chinese tallow tree existed as several varieties. Protein kinase C is a phospholipid- and calcium-dependent kinase which has been demonstrated to be the phorbol ester receptor site (NIEDEL et al., 1983 ; KjKKAWA et al., 1983) and subsequently a range of phorbol esters have been shown to activate this enzyme THE PHORBOL ESTERS

*To whom reprint requests should be addressed. 1229

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G. BROOKS et al.

(ELLIS, C ., et al ., 1985) . In this communication we describe a toxicological investigation of five distinct varieties of the Chinese tallow which are used commercially as a substitute for linseed oil, together with an assessment of the ability of isolated phorbol esters to stimulate protein kinase C. MATERIALS AND METHODS Plant material Five varieties of Sapium seb(ferum L. Roxb . were obtained from Jiangxi province in China and authenticated both in China and at the Royal Botanical Gardens, Kew, London. Spectroscopy Proton magnetic resonance ('H-NMR) spectra were obtained in CDCl, as solvent on either a Brucker WM80S (80MHz) or a VIM250 (250MHz) using TMS as internal standard ; i.r . spectra on a Perkin-Elmer 298 as surface films on KBr discs, u.v . spectra on a Perkin-Elmer 402in methanol as solvent; mass spectra(MS) on a Zab-1F by means of direct insertion at 70 eV and 200°C. High pressure liquid chromatography (HPLC) Analysis was performed with a Gilson model 302 using a Zorbax gold series 3 tan C-8 12 .5 cm x 4.6 mm column, a model 121 fluorescence detector and a water-methanol gradient as solvent (65 -100% methanol at I ml/min). Ezlraction ofplant material Crushed seeds (500 g) from each of the varieties were separately extracted in redistilled acetone at room temperature . The acetone was removed by reduced pressure distillation and the residue dissolved in water-methanol (2-8), partitioned against hexane to remove lipids and repartitioned with ether. Evaporation of the ether afforded a glassy resin. About 1 mg (accurately weighed) samples of the resins were dissolved in methanol to produce stock solutions for HPLC or in acetoneto produce stock solutions for biological analysis . Isolation of phorbol esters Ether soluble resins were initially purified by preparative thin layer chromatography (TLC) on silica gel G and the recovered residues were finally purified by partition TLC on Kieselguhr G buffered at pH 7.0 with phosphate buffer and containing 20%s diethylene glycol as stationary phase (EvANs, 19866, and references therein) . 12-O-I2-methylaminobenzoyll-4-deoxyphorbol-13-acetate (1). Sapintoxin A: yield, 7 .17 mg per kg, MS, m/z 523 (M+, 4%, CHO,N) ; u.v ., max 207,222, 252, 356 nm . ; i.r. max 3480, 1720, 1685, 1580 cm-','H-NMR, 7.84dd (J =1 .8, 7.8 Hz), 7.70d (J = 5 .05 Hz, exchangeable), 7.57s, 7.42t (J = 7 .3 Hz), 6.70d (I =7.8 Hz), 6.60t (J = 7 .8 Hz), 5.69s (exchangeable), 5 .64d (J =9 .6 Hz), 5.56d (J = 4.1 Hz), 4 .04s (2H) 3.28m, 2.93d (J = 5.05 Hz, 3H), 2.81m, 2.44m (2H, 1H), 2.18d (J=10.7 Hz), 2.13s (3H), 1.74m (3H), 1 .32s (3H), 1 .25s (3H), 1.22d (J = 5.15 Hz), 0.96d (J = 6.4 Hz, 3H) ppm. 12-O-f2-methylaminobenzoyll-4,20-dideoxy-5-hydroxyphorbol-13-acetate (2). Sapintoxin C: yield, 4.56 mg per kg, MS, m/z 523 (M+, 7%, CH)O,N); u.v ., max 207,222, 252, 356 nm .; i.r., max 3500,3400, 1725, 1685, 1630, 1580, 1520 cm - ;'H-NMR, 7 .87dd (J=8 .09, 2.04 Hz), 7.70bs (1H exchangeable, 1H), 7.43t (J=6 .98 Hz), 6.69d (J=8 .09 Hz), 6.59t (J=8 .09 Hz), 5.65d (J=9 .19 Hz), 5.61s (exchangeable), 5.32d (J-5 .15 Hz), 4 .87d (J=3 .67 Hz), 3.53m, 2.93d (J=6 .41 Hz), 2.63t (J=3 .67 Hz), 2.34m, 2.13s (3H), 1 .87s (3H), 1 .73bs (3H), 1 .66dd (J=6 .25, 9.56 Hz), 1 .29s (3H), 1 .20s (3H), 1 .08d (3=5 .52 Hz), 0.96d (J=6.25 Hz, 3H) ppm. 12-deoxyphorbol-13-benzoate (3): yield, 3.48 mg per kg, MS, m/z 452 (M+, 3%, C2,HOJ; u.v ., max 231 run; 'H-NMR, 7 .72- 7 .69m (2H), 7.55 - 7.51In (2H), 7 .37bs, 5.30s, 4.22m (2H), 3.29bs, 3.00bs, 2.2m (2H), 1 .71m (3H), 1.32s (3H), 1.25s (3H), 0.95s, 0.90d (J=6 .0 Hz, 3H) ppm. Examination of the lipidfraction for cryptic' esters Approximately 1 mg samples of the hexane soluble fraction were subjected to hydrolysis by both alkali (0.1 M KOH in dry methanol) and acid (I% perchloric acid in dry methanol). The reaction products were examined by both analytical TLC (silica gel G, ethyl acetate- chloroform 3-2) and HPLC for the presence of phorbol esters . Determination ofpro-inflammatory activity The irritant doses 50% (ID,o) of the pure phorbol esters were obtained by the mouse ear method, as previously described (EvANs and SCHMIDT, 1979), using female BALB/c white mice over the dose range 0.001 Ng/ear to a maximum of 10 Mg/ear .

Chinese Tallow Stimulates Kinase C

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Protein kinase C assay Protein kinase C was isolated from rabbit brain (PARKER et al., 1984) and assayed at 30°C using histone III-S as substrate. To a total volume of 40 MI containing 12.5 mM magnesiumchloride, 1 .5 mM calcium chloride, 1 .25 mg/ml of histone, IMP]-ATP, 0.03 mg/ml of phosphatidylserine and 60 pmoles to 500 nmoles of phorbol ester, 5p1 of enzyme were added. The reaction was terminated after 10 min by spotting 25 pl of the assay mixture onto phosphocellulose paper (Whatman). The strips of paper were rinsed and incorporation of 'P into protein assessed by Cerenkov counting. RESULTS AND DISCUSSION

Examples of the genus Sapium indigenous to China include four separate species (PAX and HOFFMAN, 1934), of which Sapium sebiferum L. Roxb. (the Chinese tallow tree) is the most widely used. This plant is the source of a drying oil which is used as a substitute for linseed oil. No varieties of this species have previously been recorded, but we were able to obtain distinctly different morphological types directly from China, all of which are available under the name Chinese tallow seeds. The five varieties used in this study were all authenticated as Sapium sebiferum at Kew gardens in London. The varieties varied in their size and shape due to different thicknesses of the characteristic tallow layer (Table 1), but the appearance of the inner seeds were essentially similar. These types are known by a number of local names (Table 1) and have possibly arisen due to the extensive geographical range of the plant in China. However, the prepared tallow or oil of these seeds would not be assignable to any one particular type and we have therefore analysed the oil of all varieties for the presence of phorbol esters (Fig . 1). Of the five samples investigated, four were found to contain phorbol esters and two varieties contained sufficient quantities for isolation and structure determination of the major components . The oils from seeds of plants from the family Euphorbiaceae are known to produce `cryptic' phorbol esters . These compounds are acylated at the C-20 position of the tigliane nucleus and are not biologically active, but may be converted to the active forms by means of lipase activity (EVANS, 1986x) . The five samples of seed oil examined here were investigated for the presence of such compounds by attempted hydrolysis of the lipid fraction, but no evidence for this type of compound was obtained . Three phorbol esters were isolated from the polar fractions and their structures determined by spectroscopic methods. These were identified as sapintoxins A and C, previously known from the fruits of Sapium indicum (TAYLOR et al., 1981a,b), together with the new natural product 12-deoxyphorbol benzoate. These pure compounds were examined for their pro-inflammatory activity on mouse skin using an established assay (EVANS and SCHMIDT, 1979). Sapintoxin A and 12-deoxyphorbol benzoate elicited inflammation of mouse skin with ID5o values of 0.042 and 2.60 nmoles/5 P1 per ear, respectively, whilst sapintoxin C was inactive in doses of up to 20 nmoles/5 0. This is in accordance with previous results using phorbol esters, in that 20-deoxyphorbol derivatives Sapium sebiferuln Tallow/whole seed (% w/w)

TABLE 1 . VARIETIES OF

Local name

AND THEIR PHORBOL ESTER CONSTITUENTS

Average size of seeds (n=10) (mm) Not known 35 .6 5.21 x 7 .58 Grape sapium 28 .2 6.14 x 7.62 Chicken-foot 41 .5 6.47 x 11 .15 Dog-trail 33 .6 5.84 x 8.93 Wooden-club 36 .7 5 .95 x 8.74 1 =sapintoxin A; 2=sapintoxin C; 3= 12-deoxyphorbol 13-benzoate .

Phorbal esters detected None l, 2,3 1 Trace 1 Trace 1

G . BROOKS et al.

123 2

le H3C

FIG . 1 . THE STRUCTURES AND BIOLOGICAL ACI7vmEs OF ISOLATED PHORBOL ESTERS . Compound R R R R Ig, SD R 1 H H 0 .042nmoles 0.11 76 nM 2-methylaminoacetate OH benzoate 2 acetate H H >20 nmoles 2-methylaminoOH >100 nM benzoate 3 benzoate OH H OH 2 .6 nmoles 0 .01 176 nM H 1=sapintoxin A ; 2=sapintoxin C ; 3=12-deoxyphorbol 13-benzoate . In =dose producing inflammation of mouse skin in 50% of the test animals following application of 5 pl of phorbol ester calculated by PROBIT analysis ; Ka =the concentration at which 504 activation of protein kinase C occurs (12-O-tetradecanoylphorbol 13-acetate used as standard) . were inactive in a number of

1987).

in vitro

and

in vivo

test systems (EvANS and EDWARDS,

Phorbol esters are known to substitute for diacylglycerol in the activation of protein kinase C (NIEDEL et al., 1983 ; KIKKAWA et al., 1983) and membrane bound forms of that enzyme are believed to be the phorbol ester receptor site . The activation of protein kinase C by phorbol esters may be involved in several biological roles ranging from tumour promotion to inflammation (AITKEN, 1986). Accordingly, we have examined the abilities of the phorbol esters from the Chinese tallow tree to stimulate protein kinase C in a cell free assay (Fig . 1) . Both of the phorbol esters which demonstrated pro-inflammatory activity in vivo significantly activated protein kinase C, with K, values of 76 and 176 nM, respectively. Once again, the 20-deoxyphorbol derivative was inactive up to a concentration of 1001AM (Fig . 1 .) . Of the five varieties of Chinese tallow available to us, four contained significant quantities of phorbol esters . These compounds are potent pro-inflammatory agents and were shown to induce significant activation of protein kinase C . The variety known as grape sapium was the most potent, in that all three phorbol derivatives were detected in isolatable quantities . Sapium sebiferum originating from China may therefore be derived from a species which has evolved various local morphological and chemical varieties . Attention needs to be given to the source of imported drying oils for inclusion in manufactured products if both pro-inflammatory and tumour-promoting toxic effects are to be avoided .

Acknowledgements- We are grateful to Prof. CHEN CAISHUI for providing samples of Chinese tallow tree seeds and to the Herbarium at Kew for authenication of these samples as Sapium sebijerum . This work was supported by an S .E .R .C project grant (F .J .E . and A.A .), two S .E .R .C . studentships (C .E. and N .A .M .) and a Pharmaceutical Society studentship (G.B.) . REFERENCES AITxEN, A . (1986) The biochemical mechanism of action of phorbol esters . In : Naturally Occurring Phorbol Esters. Chap . 10, p . 271 (EvANs, F . J ., Ed .). Boca Raton, U .S .A . : CRC Press Inc .

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ELLIS, C., MORRICE, N., AtrKEN, A., PARKER, P. J . and EvANS, F. J . (1985) Activation of protein kinase C by tumour promoting and non-promoting phorbol esters. J. Pharm. Pharmac. 37, 23P. E, m, M. D. (1978) Dangerous plants, snakes, arthropods and marine life . In: Toxicity and Treatment, p . 39 (ELLIS, M. D., Ed .). Hamilton, IL : Drug Intelligence Publications Inc. EvANS, F. J . (1986x) Environmental hazards of diterpene esters from plants . In: Naturally Occurring Phorbol Esters, Chap . 1, p. 1 (EvANs, F. J., Ed .) . Boca Raton, U.S.A .: CRC Press Inc. EvANS, F. J. (1986b) Phorbol: its esters and derivatives . In: Naturally occurring phorbol esters, Chap. 7, p. 171 (EvANs, F . J., Ed .). Boca Raton, U.S .A . : CRC Press Inc. EvANs, F. J. and EDwARDs, M. C. (1987) Activity correlations in the phorbol ester series . Bot . J. Linn . Soc . 94, 231 . EvANs, F. J . and ScHMIDT, R. J. (1979) An assay procedure forthe comparative irritanty testing of esters of the tigliane and daphnane series. Inflammation 3, 215. IüKKAWA, U., TAKAi, Y., TANAKA, Y., MYAKE, R. and NISHIZUKA, Y. (1983) Protein kinase C as a possible receptor protein of tumour promoting phorbol esters . J. biol. Chem . 258, 11442. NIEDEL, J. E., KUHN, L. J. and VANDENBARK, G. R. (1983) Phorbol diester receptor co-purifies with protein kinase C . Proc. natn. Acad. Sci. U.S.A . 30, 36 . OHIGASHI, H., AHrsuvA, T., HIROrA, M., Kosmmizu, K., ToKuDA, H. and ITo, Y . (1983) Tigliane type diterpene esters with Epstein-Barr virus-inducing activity from Sapium sebjferum . Agric. biol. Chem . 47, 1617 . PARKER, P. J ., STAaEL, S. and WATERFiELD, M. D. (1984) Purification of protein kinase C from bovine brain to homogeneity, identity with the phorbol ester receptor. Embo. J. 3, 953. PAx, F. and HoFFmAm, H. (1931) Euphorbiaceae. In: Die Naturlichen Pflanzenfamifen, 2nd Edn, 19C, p. 208 (ENOLER, A. and PRANTL, K., Eds) . Leipzig: Englemann. RADcLIFt"S3zSArrrH, A. (1986) A review of the family Euphorbiaceae. In : Naturally Occurring Phorbol Esters, Chap . 3, p. 63 (EvANS, F. J., Ed .). Boca Raton, U.S .A . : CRC Press Inc. Smp, E. H., OTT, H. H. and HEcKER, E. (1983) Skin irritants and tumour promoting diterpene esters of the tigliane type from the Chinese tallow tree. Planta med. 49, 199. TAYLOR, S. E., GAFUR, M. A., CHOUDHURY, A. R. and EvANs, F. J. (1981x) Sapintoxin A, a new biologically active nitrogen containing phorbol ester. Experientia 37, 681. TAYLOR, S. E., GAFUR, M. A., CHOUDHURY, A. R. and EvANS, F. J. (1981b) Nitrogen containing phorbol derivatives from Sapium indicum . Phytochemistry 20, 2749 .