Plants as sources of antimalarial drugs, part 6: Activities of Simarouba amara fruits

.Townalof E~hno~~~~o~g~, Elsevier Scientific Publishers

22 (1938) 133- 190 Ireland Ltd.

183

PLANTS AS SOURCES OF ANTI~A~RIAL DRUGS, PART 6: ACTIVITIES OF SIMAROU??A AMARA FRUITS

MELANIE J. O’NEILL’, DOROTHY H. BRAP’, PETER BOARDMAN*, COLIN W. WRIGBT”, J. DAVID PHILLIPSONa~ DAVID C. WARHURST&, MAHA3IR P. GUPTA’, M. CORREYAc and P. SOLISC %yartment of Pha~~o~os~, The School ofPha~~~, ~niveTs~t~ of London, 29-39, Bmozswick SquaTe,London, WClNlAX, ~~e~Ttrnent ofMed~calProtozoology, LondonSchool of Hygiene and TropicalMedicine, Keppel Stree t, Lo&o% WClE YHTIU.K.Iad %s tafe ta Un~vers~~u~ ~~e~ub~~~ of Pa~rn~ (Accepted December 9.1987)

Summary Extracts prepared from Simarouba amaru fruits collected in Panama have been found to be active against P~srnoa~~rn falciparum in vitro and against P~smod~urn berghea’ in mice. Four active quassinoids have been identified as ailanthinone, 2’-aeetylglaucarubinone, glaucarubinone and holacanthone,

Introduction

The urgent requirement for novel antiplasmodial agents which are active against chloroquine-resistant strains of Plasmodium species, particularly P. ~~~~p~~rn, has prompted the scrutiny of some natural traditional remedies (Guru et al., 1983; Khalid et al., 1986; Klayman, 1985; Monjour et al., 1987; O’Neill et al., 1985; Pavanand et al., 1986; Trager and Polonsky, 1981). Some species of the family Simaroubaeeae, which are used in traditional medicine to treat malaria (Morton, 1981; Steck, 19721 have been shown to possess high antiplasmodial activity which can be attributed to their quassinoid constituents (Ghan et al., 1986; Guru et al,, 1983; Trager and Polonsky, 1981). It has also been found that the in vitro anti-P. fa~~~pa~rn activity of quassinoids does not always parallel their in vitro cytotoxicity to some mammalian cells @‘Neil1 et al., 1986). As part of a study of the antimalarial properties of quassinoids and simaroubaceous plants we have examined S~rn~To~ba arnara fruits, extracts of which have been found previously to be active against avian malarias (Spencer et al., 1947). The related species S. glauca, is used traditionally in Central America to treat malaria (Morton, 1981). 0378-87~1~~~03.15 0 1988 Elsevier Published and Printed in Ireland

Scientific

Publishers

Ireland

Ltd.

184

R

Yield f% of fruit weight)

Ailanthionine

(1)

2’-Acetylglaucarubinone Holacanthone Glaucarubinone

T(Me)H (2)

(3) (4)

T(Me)

3%H, TH, (OAcP’CH

2 TH,

0.006 0.007

2’CH,

0.019

*‘C(Me) (OHV’CH, 4%H,

0.033

Fig. 1. Quassinoids from S. amara fruits.

Materials and methods

Plant material 8. umara fruits were collected in the Colon region of the Republic of Panama and were authenticated in the Department of Botany, University of Panama, where herbarium specimens are maintained. The dried fruits were transported to London for extraction, Preparation

and PuT~f~cut~o~of extract

Powdered dried fruits (200 gf were defatted with petroleum ether and then exhaustively extracted at room temperature with methanol followed by water. The concentrated methanolic extract (38 g viscous liquid) was chloroform, butanol and water. The concentrated partitioned between chloroform extract (3 g syrupy residue1 was chromatographed over with a hexanelchloroformlmethanol polyamide (approx. 300 g). Elution gradient produced 9 fractions (A-1). The fractions were purified by thinlayer chromatography over silica gel GF254 developed in chloroform/ isopropanol (9:ll or chloroform/methanol (8:21 and visualised by viewing under UV light or by spraying either with 5% phosphomolybdic acid or 60°h sulphuric acid. Final purification was achieved by high performance liquid ehromato~aphy using an Ultrasphere ODS 5 column eluted with a methanols

185

water gradient and monitored at 254 nm. At each stage in the purification, fractions were examined for in vitro activity against P. falciparum. column yielded ailanthinone (1) (12.4 mg), 2’The polyamide acetylglaucarubinone (2) (14.8 mg) and holacanthone (3) (5 mg) from fraction B, glaucarubinone (4) (17.8 mg) and holacanthone (20 mg) from fraction C, glaucarubinone (47.3 mg) and holacanthone (12.7 mg) from fraction D. Identification

of quassinoids

Proton magnetic resonance spectra were determined in CDCl, on a Brucker WM-250 MHz instrument. EIMS were obtained using a VG 12-250 Quodrupole mass spectrometer. UV spectra were obtained on a Perkin Elmer UV Visible 402 spectrophotometer. AiZanthinone

(1)

UV Amax(MeOH) 240 nm; PMR (CDCl,) d 0.97 (3H,t,J = 7.4 Hz,Me-4’1, 1.11 (3H,d,J = 6.8 Hz,Me-2’1, 1.19 (3H,d,J = 7.0 Hz,Me-131, 1.21 (3H,s,Me-101, 1.49 - 1.85 (2H,m,Cl&-3’1, 2.03 (3H,s,Me-41, 2.35 (2H,m,H-13,H-14). 2.43 (H,m,I-N!‘), 2.75 (H,s,H-91, 2.99 (H,br.d,J = 11.4 Hz,H-5), 3.59 (H,br.s,H-121, 3.69 (H,d,J = 8.9 Hz,CH,-81, 3.97 (H,d,J = 8.9 Hz,CH,-8), 4.08 (H,s,H-11, 4.64 (H,br.s,H-71, 5.59 (H,d,J = 11.0 Hz,H-15) 6.16 (H,br.s,H-3); EIMS m/e (rel.int.1 478 (28”/6)M + , 460(15O/o), 394(18O/o), 376(33%), 358(32O/o), 319(21%), 247(37%), 201(22%1), 151(16%), 135(17%), 94(23%), 85(300/o), 74(44%), 57(100%), 41(54%). The identity of (1) was confirmed by comparison with an authentic sample and literature values (Waterman and Ampofo, 1984). 2’Ace tylglaucarubinone

(2)

UV A,,, (MeOH) 240 nm; PMR (CDCl,) d 0.98 (3H,t,J = 7.5 Hz,Me-4’1, 1.22 (3H,s,Me-101, 1.28 (3H,d,J = 7.1 Hz,Me-131, 1.62 (3H,s,Me-2’1, 1.85-2.55 (3H,m,CH,-3’,H-141, 2.45 (H,m,H-13) 2.64 (H,s,H-91, 3.07 (H,br.d,J = 11.9 Hz&5), 3.61 (H,d,J = 3.4 Hz,H-121, 3.71 (H,d,J = 9.0 Hz,CH,-81, 3.97 (H,d,J = 8.9 Hz,CH,-81, 4.05 (H,s,H-11, 4.77 (H,br.s,H-7), 5.16 (H,d,J = 10.7 Hz&l-15) 6.15 (H,br.s,H-3); EIMS m/e (rel.int.1 536(6%)M + , 518(5%), 376(68%L 358(53%), 270(300/o), 255(280/o), 239(26%), 225(39%), 213(34%), 201(30%), 115(33%), 100(29%), 43(100%). The identity of (2) was confirmed by comparison with an authentic sample and literature values (Waterman and Ampofo, 1984). Holucanthone

(3)

UV Amax(MeOH) 240 nm; PMR (CDCl,) d 1.12 (3H,d,J = 7.1 Hz,Me-131, 1.21 (3H,s,Me-101, 2.03 (3H,s,Me-41, 2.16 (3H,s,Me-2’1, 2.77 (H,s,H-91, 2.96 (H,br.d,J = 12.6 Hz,H-5), 3.59 (H,br.s,H-121, 3.70 (H,d,J = 9.0 Hz,CH,-8), 3.97 (Kbr.&J = 9.0 Hz,CH,-81, 4.10 (H,s,H-11, 4.62 (H,dd,J = 2.9,2.4 Hz,H-71, 5.65 (H,d,J = 11.6 Hz,H-15). 6.16 (H,q,J = 1.2 Hz, H-3); EIMS m/e (rel.int.1 436(21%)M + I 394(4%), 377(80/b), 319(80/b), 247(20%), 229(13%), 151(38%), 135(17%), 91(25%), 69(200/c), 55(210/c), 43(100%). The identity of (3) was confirmed by comparison with an authentic standard and literature values (Wall and Wani, 1970).

186

UV Amax (MeOH) 240 nm; PMR (CDCI,l d 0.97 (3H,t,J = 7.5 Hz,Me-4’1, 1.14 (3H,d,J = 7.7 Hz,Me-131, 1.22 (3H,s,Me-101, 1.46 (3Hs,Me2’1, 2.0-2.5 12H,m,CH,-3’1,2.03 (3H,s,Me-41 2.73 (H,s,H-91, 3.00 (H,br.d,J = 12.7 Hz,H-51, 3.59 (H,br.s,H-121, 3.71 (H,d,J = 9.0 Hz&H,-81, 3.98 (H,d,J = 9.0 Hz,CH,-81, 4.07 (H,s,H-11,4.67 (H,br.s,H-71, 5.63 (H,d,J = 11.1 H.z,H-1516.17 (H,q,J = 1.4 Hz,H-3); EIMS m/e (rel.int.1 494(22%lM -t , 476(31%1, 458(11%1, 447(12*/o), 376(90~), 185(15%1, 151~37O~),135(97*/o), 73(1000,01, 55(90%), 43(85O~). The identity of (41 was confirmed by comparison with an authentic standard and literature values (Waterman and Ampofo, 19841.

Extracts and quassinoids were assayed for activity against a muitidrug resistant strain (E(l) of P. faleipmwn (Thaithong et al., 1983) in vitro. The technique, based on that described by Desjardins et al. (1979) measures inhibition of incorporation of [3H]hypoxanthine, into the parasite. Precise details of the method used are given by O’Neill et al. (1985, 19861.

Extracts and quassinoids were monitored for in vivo antimalarial activity using a d-day suppressive test, based on that described by Peters 09801 against P. berghei, strain N infections in mice. Oral dosing with plant extracts and quassinoids was used throughout. Mice were randomly grouped and 5 mice were used at each dose. Each mouse received a total of 4 doses given over 4 days. Each plant extract or quassinoid was tested at 4 concentrations. Five water-dosed mice were used as the control. Precise details of this procedure are given by O’Neill et al. (19871. Results and discussion In vitro activity

aguiwt

P. fal~~~u~rn

Of the original three crude extracts which were assessed for in vitro activity, the methanolic extract, having an IC, value of approx. 0.5 pg ml-’ (Table 11, proved to be most active. Further partition of the methanoIic extract resulted in the concentration of the in vitro activity in the chloroform phase (IC,,, 0.05 pg ml-‘). Purification of this chloroformic phase by column chromatography yielded 9 fractions, of which all except the first exhibited some activity against P. fdciparum in vitro. The most active of these fractions, B-F, were found to be rich in bitter constituents, which were identified as quassinoids. Four quassinoids were eharacterised by their spectroscopic (UV, PMR, EIMSl parameters as ailanthinone (11, 2’-acetylglaucarubinone (21, holacanthone (31 and glauearubinone (41. Each of these quassinoids has been

187 TABLE 1 IN VITRO ANTIMALARIAL ACTIVITY (INHIBITION OF INCORPORATION [8H]HYPOXANTHINEl AGAINST I? FALCIPARUM (Kll OF CRUDE EXTRACTS ISOLATED QUASSINOIDS FROM S. AMARA FRUITS Extracts

IC, (pg rnl-lp

Petroleum ether Methanol Chloroform Butanol Aqueous Aqueous

500 0.5 0.05 0.5 5 5

Quassinoids

IC, (ng mW

Ailanthinone 2’-Acetylglaucarubinone Holacanthone Glaucarubinone

9 8 7 4

Chloroquine diphosphate

OF AND

IC,, (nM1

(7-ilk (6-11) (4- 13) 12-71

14 16 8

210 (190 - 2401

407

‘Based upon lo-fold dilutions in duplicate. bBased upon 2-fold dilutions in duplicate. 95% confidence interval given in parentheses.

isolated from simaroubaceous plants previously (Polonsky, 1985) and the most abundant compound, glaucarubinone, and its 2’-acetyl derivative have been detected earlier in S. amara fruits. However, this is the first report of the occurrence of ailanthinone and holacanthone in S. amara fruits. The activity of the quassinoids against P. falciparum in vitro was determined using 2-fold dilutions in duplicate and as shown in Table 1, they were found to be some 23- 52 times more active than chloroquine. The variation in the antiplasmodial activities of the quassinoids is attributable TABLE 2 IN VITRO ANTIPLASMODIAL QUASSINOIDS

ACTIVITY

COMPARED

A P. falciparum

IC, (ng ml11 Ailanthinone 2’-Acetylglaucarubinone Holacanthone Glaucarubinone ‘From Cassady and Suffness (19801.

9 8 7 4

TO IN VITRO CYTOTOXICITY

OF

B” KB cells ED, (ng ml-l)

B/A

30 5 200 40

3.3 0.6 29 10

188

solely to their substituents at C-15, an effect which has been noted previously for quassinoids from other Simaroubaceae species (O’Neill et al., 1986, 19871. As observed for other quassinoids (O’Neill et al., 19861, in vitro antiplasmodial activity for the four compounds of this study does not follow cytotoxicity. Table 2 lists the comparative toxicity to KB cells (human epidermoid carcinoma of the mouth) of the quassinoids isolated from S. umara fruits. Overall, 2’-acetylglaucarubinone has the least favourable antiplasmodial to cytotoxicity ratio of 0.6, whereas holacanthone has a much more favourable ratio of 29. These data suggest that for quassinoids, antiplasmodial activity and toxicity to mammalian cells may be diverging properties. In vivo activity against P. berghei

Four of the crude extracts of the fruits were tested for activity against P. strain N and the results are given in Table 3. Accurate IC,, values

berghei

TABLE

3

SUPPRESSION OF PARASITAEMIA IN P. BERGHEI (N) INFECTED AND QUASSINOIDS FROM S. AMARA FRUITS Extracts

Percentage inhibition at highest dose used (mg kg-’ day-‘)’

Petroleum ether Methanol Chloroform Butanol Aqueous Aqueous

N.T. N.T. 40 at 50 at 30 at 30 at

Quassinoids

ED, (mg kg-’ day-‘)

Ailanthinone

1.25(0.90 - 1.75)

2’.Acetylglaucarubinone Holacanthone Glaucarubinone

2.19(1.62-2.95)

Chloroquine diphosphate

deathsb

0 0 0 0

1.91) 1.26)

2.27(1.83-2.81)

“Oral dose given over 4 days. b5 mice at each dose. cExtrapoiated result.

BY EXTRACTS

Toxic

600 900 900 900

1.70(1.530.86(0.59-

MICE

ED, (mg kg-’ day-‘) 4.13(2.96-5.75) 11.62(8.624.50(4.033.43(2.35-

15.66)’ 5.03) 5.02)

6.02t4.86 - 7.46)

5 at 9 1 at 3 0 at 9 1 5 0 0

at at at at

18 9 3 30

189

were not obtained, but each of the extracts produced inhibition of between 30% and 50% at the highest doses (600 or 900 mg kg-’ day-‘) used. These results lend some support to the traditional use of S. amara fruits as a remedy for malaria. None of the extracts induced any deaths at the highest doses given. The chloroformic extract produced around 4OW reduction of parasitaemia at a dose level of 600 mg kg-’ day-l, Since 3 g of chloroformic extract gave a total yield of quassinoids of about 130 mg, 600 mg of the extract can be assumed to have contained about 25 mg of quassinoids. The ED, values for the quassinoids ranged from 0.86 to 2.19 mg kg-’ day-’ as shown in Table 3, and thus the activity displayed by the total chloroformic extract is somewhat lower than would be expected, based upon its quassinoid constituents. It can be inferred from this that the chloroformic extract contains, in addition to its quassinoids, constituents which may antagonise the antiplasmodial activity of the quassinoids. 2’-Acetylglaucarubinone showed the least activity of the four quassinoids. The highest concentration tested was 9 mg kg-’ day-’ and thus the ED, value of 11.62 mg kg-’ day-’ given in Table 3 is an extrapolated result. Glaucarubinone appeared to be the most active quassinoid having an ED, value of 3.43 mg kg-’ day-‘. It was also a highly toxic compound, being 100% lethal at 9 mg kg-’ day-‘. Recently, other workers have reported (Monjour et al., 19871 that glaucarubinone given either orally or peritoneally to mice, is too toxic to be of value as an antimalarial agent. Ailanthinone and holacanthone had similar ED, values of 4.13 and 4.50 mg kg-’ day-‘, respectively. However, whereas ailanthinone showed high toxicity, killing all 5 mice at 9 mg kg-’ day-‘, holacanthone showed relatively a lower toxicity, causing only 1 death out of 5 mice at 18 mg kg-’ day-‘. These findings reinforce the results obtained in vitro and lend support to the conclusion that the antimalarial activities and general toxicities of the quassinoids do not go hand in hand. The potential value as an antimalarial of holacanthone in particular and of quassinoids in general clearly merits further investigation. Acknowledgements We are grateful for financial support to the Commission of the European Communities Directorate General for Science Research and Development and to the Medical Research Council. References Cassady, J.M. and Suffness, M. (1980) In: J.M. Cassady

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