Plcinta Med. 57(1991) 341

Antimalarial Activity of Tanzanian Plants and their Active Constituents: The Genus Uvaria1 M. H. H. Nkunya2'3, H. WeenenZ4, D. H. Bray5, Q. A. Mgani2, andL. B. Mwasumbi6 'Part 3 in the series Antimalarial activity of Tanzanian plants. For parts 1 and 2, see: (3) and (4) 2 Department of Chemistry, University of Dares Salaam, P. 0. Box 35061, Dares Salaam, Tanzania Address for correspondence Present address: Quest International, P. 0. Box 2,1400 CA Bussum, The Netherlands 6

Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, U.K. Department of Botany, University of Dar des Salaam, P. 0. Box 35060, Dares Salaam, Tanzania

Received: May 18, 1990

ether, dichloromethane, and methanol extracts of nine Uvaria species found in Tanzania, as well as of seventeen compounds isolated from these extracts.

Petroleum ether, dichloromethane, and methanol extracts of leaves, stem, and root bark of nine Uvaria species: U dependens, U. faulknerae, U kirkii, U leptocladon, U lucida ssp. lucida, Uvaria sp. (Pande),

U scheffleri, and U tanzaniae were tested for their in vitro activity against the multidrug resistant Ki strain of Plasmodiumfalciparum. The IC,0 values of the extracts

varied between 5 and 500 tg/ml. The most active extracts were obtained from the stem and root bark of U lucida ssp. lucida and Uvaria sp. (Pande) and the root bark of U scheffleri, all of which had IC,0 values between 5 and 9 .tg/ml. Among the compounds isolated, uvaretin, diuvaretin, and (8' ,9 '-dihydroxy)-3-farnesylindole were the most active (IC50 = 3.49, 4.20, and 2.86 tg/ml, respectively).

Key words Uvaria species, Annonaceae, antimalarial activity, dihydrochalcones, sesquiterpenoids.

Introduction Malaria is one of the most prevalent and dangerous diseases in the tropics and subtropics. It is estimated that in 1983 there were about 200 million malaria cases in Africa alone (1). Moreover, malaria accounts for an infant mortality of about 1 million annually (1).

Of the four most common human Plasinodium species, P. falciparum causes about 80% of all cases of the disease, mainly in tropical Africa (2). Because

the parasites are developing resistance to most of the aminoquinoline-based and sulfa drugs which are presently in use, efforts are now being directed in obtaining drugs which have different structural features, either synthetically or from plants (3—9).

Uvaria species have been demonstrated to be a source of many new compounds, some of which have a wide range of biological activities (10). In this paper we report on the antimalarial activity of the crude petroleum

Materials and Methods Plant materials Nine Uvaria species were collected from different parts of Tanzania (Table 1) and identified at the Herbarium, Department of Botany, University of Dar es Salaam, where voucher

specimens are deposited. The description of the new Uvaria species (Pande) is in progress.

Extraction and isolation procedures Air dried, pulverized root bark, stem bark, and leaves were extracted as previously described (3). The concentrated crude extracts were then tested for their in vitro antimalarial activity using the multidrug resistant Ki strain of P.falciparum (see below). The most active extract from each plant was fractionated by chromatography to give crude compounds which were purified further by repeated column chromatography and by gel filtration [Fractogel PVA 500 (Merck) or Sephadex LH-20 (Pharmacia)] and! or preparative TLC (normal or silanized silica gel). Crystalline compounds were recrystallized from suitable solvents. The compounds were then identified on basis of their chemical, spectral, and other physical properties and, if necessary and possible, new structures were confirmed by X-ray crystallography. The compounds were then tested for their antimalarial activity.

Antimalarial testing The in vitro antimalarial testing of the crude ex-

tracts and pure compounds were carried out as previously described (3, 4, 5, 9, 11). For comparison purposes the methanolextract of a Cinchona bark (grown in Usambara, Tanzania) was tested (IC50 = 0.50 ig/ml). Similarly, the activity of chloroquine diphosphate and quinine hydrochloride were also determined and their IC,0 values were 0.5 16 and 0.038 .tg!ml, respectively.

Results and Discussion All nine Uvaria species which were investigated showed some activity against P. falciparum malaria parasites, their IC50 values varying between 5 and 500 .tg/ ml. Extracts from the root and stem bark of U lucida ssp.

lucida and Uvaria sp. (Pande) and the root bark of U scheffleri were the most active, with IC,0 values between 5

and 9 j.tg/ml. In all plants studied, leaf extracts showed lower activity than stem and root bark extracts (Table 1).

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Abstract

342 Planta Med. 57(1997)

M. H. H. Nkunyaetal. Table 2 AntimalariaI activity of compounds f rom Uvaria species.

Place

Part Collecteda Used

Nameofplant

U. dependens Eng. & Diels

2

U. fau/knerae Verdc.

3 4

U Kirkii Hook. f.

5

U.IeptocladonOliv.

U.iucidassp./ucidaBenth.

Uvariasp.(Pande)

67,8

4

Root bark stem bark leaves Root bark stem bark leaves Root bark stem bark leaves

Rootbark stem bark leaves Root bark stem bark leaves Root bark stem bark leaves

U. pandensis Verdc.

4

Rootbark stem bark leaves

U. scheffleriDiels

9

Root bark stem bark leaves

UtanzaniaeVerdc.

10

Rootbark stem bark

Antimalarial Activity(3,4)1' P.E. CH2CI2 MeOH

Compound

+++ + + +++ —

Uvaretin (1)

U faulknerae, U. kirkii, U Iucidassp. lucida, U. leptocladon, Uvaria sp. (Pande), U. tanzaniae

Diuvaretin (2)

U. kirki U. lucida

Source



+ +

+ +



+

++ ++

— —

+ —

+++ ++ +++ +++ —

ssp.





+



+++ +++ ++ +++ +++ —

+

+ +

sp.

Triuvaretin (3) lsotriuvaretin (4) Chamuvaretin (5)

+++ ++++ ++

+++ ++++ ++++

++ +++

++++ +++ ++++ +++ + +

++ ++ ++

++ +++ +

+ +

+

+



+

++ ++++ ++++

++ ++ ++ ++

+ +

++ +++ ++ +++ +++ ++

= Gombe National Park; 2 = Kwamkoro, Amani; 3 = Boza, Pangani; 4 = Pande forest; 5 Fundi Mt., Korogwe; 6 = Chalinze; 7 = Bushiri, Pangani; 8 = Kwamkono, Handeni; 9 = Soni, Lushoto and 10 = Longuza Forest, Amani. Antimalarial activities are given in IC50 values and these have been categorized as follows: ++±±: IC50 = 5 to 9 pg/mI; +++: IC50 = 10 to 49 tg/ml; 4±: IC50 = 50 to 99 g/ml; +: IC50 = 100 to 499 pg/mI; —: IC50 >499 pg/mI (inactive).

IC50

(pg/mI)

Uvaretin trimethyl ether(6)a Chamanetin (18) Lucidene(7) Tanzanene(8) Guaiol (9) 3-Farnesylindole (10)

lucida, Uvaria

3.49

(2.29—5.32)

4.20

(3.22—5.45)

(Pande)

U. leptocladon U. leptocladon, U. tanzaniae U. leptocladon U tanzaniae, U. lucida ssp. lucida

46.02 20.85

U. faulknerae U. lucida ssp.lucida

45.52

(3.68—7.73)

8.31

(6.78—10.18) (38.38—53.98)

50 50

Uvariasp. (Pande) U. pandensis

16.7

Upandensis

(37.74—55.49) (15.33—27.35)

5.32

Utanzaniae

(6',7'.Dihydro.8', 9'.dihydroxy). 3.farnesindole(11)

95% confidence intervals (pg/mi)

>50

5O

(8',9'-Dihydroxy).

3-farnesylindole (12) (+)-Pandoxide (13)

U pandensis

U. pandensis (±)./3.Senepoxide (14) U. pandensis,

(—).Pipoxide (15)

2.86

> 25

U.faulknerae

>25

U. pandensis

8.35

7-Hydroxy.4',5,6,8tetramethoxy. U.pandensis flavanone(16) U. scheffleri Glutenol (17) Chloroquine — diphosphateb quinine hydrochlorideb_

(2.35—3.45)

(6.54—10.65)

>50 > 50 0.516 0.038

(0.417—1.289) (0.038—0.058)__—

Obtained by methylation of 1. Included as standards.

Inmost cases the highest antimalarial activ-

as compared to 2 (IC50 = 4.20 .sg/ml). However, these varia-

dichloromethane extracts. The only exceptions to this observation were the methanol extracts of the stem bark of U. lucida ssp. lucida and the root bark of U scheffleri, which showed IC50 values between 5 to 9 g/ml. We have isolated

significant. This suggests that apart from the hydroxyl

ity was found in the petroleum ether (P. E.) and tions in activity between I and 6, and 2 and 5 are not very

several compounds from the most active fractions and tested their antimalarial activity against the multi-drug resistant Ki strain of P. falciparum. Table 2 gives the antimalarial activity of the tested compounds.

It can be noted from Table 2 that the most active compounds are C-benzylated dihydrochalcones (the

uvaretins) and indolosesquiterpenes. Since there are no characteristic structural similarities between these two classes of compounds, we assume that they may act diffe-

groups, other structural features are necessary for the observed antimalarial activity of the dihydrochalcones, besides molecular size.

Uvaretin and diuvaretin are the most widely occurring dihydrochalcones among Uvaria species. The two compounds have been isolated from U accuminata (13), U angolensis (14), U chamae (15), U kirkii (16, 17), U lucida ssp. lucida (18), U. tanzaniae (19), Ufaulknerae, U leptocladon (12), and Uvaria sp. (Pande) (Table 2). Uvaretin and diuvaretin also show cytotoxic and antibacterial activities (16, 20).

rently on malaria parasites. The activity of the uvaretins de-

creases with increasing molecular size. Thus there is a progressive decrease in activity from uvaretin (1), diuvare-

tin (2), and triuvaretin (3) (12) (IC50 = 3.49, 4.20, and 46.02 .tg/ml, respectively). The phenolic hydroxyl groups also seem to contribute to good activity, as shown by the de-

crease in activity in uvaretin trimethyl ether (6) (IC50 = 8.31 .tg/ml) as compared to the unmethylated compound (IC50) 3.49 .tg/ml) and chamuvaretin (5) (IC50 = 5.32 .tg/ml)

An interesting observation was noticed among the indolosequiterpenes 10—12 isolated from U pandensis (21, 22). The activity of these compounds appears to be largely determined by the sesquiterpene side chain and not the indole moiety. Thus, whereas 3-farnesylindole (10) is weakly active (IC50 = 16.7 j.tg/ml), the 8',9'-dihydroxyl isomer (12) is much more active (IC50 = 2.86 sg/ml). This activity depends on the presence of the C-

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Table 1 Antimalarial activ ity of some Uvaria species.

AntimalarialActivity of Tanzanian Plants and their Active Constituents: The Genus Uvaria

References 1 UNDP/World Bank/WHO (1985) Special programme for research 2

Both the novel benzopyranyl sesquiterpenes, lucidene (7) and tanzanene (8), isolated from U

Weenen, H., Nkunya, M. H. H., Bray, D. H., Mwasumbi, L. B., Kinabo, L. S., Kilimali, V.A. F. B. (1990) Planta Med. 56, 368. Weenen, H., Nkunya, M. H. H., Bray, D. H., Mwasumbi, L. B., Kinabo, L. S., Kilimali, V.A. F. B., Wijnberg, J. B. P. A. (1990) Planta Med. 56,

lucida ssp. lucida (18) and U tanzaniae (19), respectively, are virtually inactive. Of the three cyclohexene epoxides, (+)-pandoxide (13), (-f)-fl-senepoxide (14), and (—)-pipoxide (15), isolated from U. pandensis (23) [(+)-/3-senepoxide was

also isolated from U. faulknerae} only (—)-pipoxide is weakly active. These compounds are electrophilic species which can be expected to react with nucleophilic centres in nucleic acids of the parasites in a Michael-type addition (4), with either elimination of the C-3 substituent or with opening of the epoxide at C-6. (—)-Pipoxide, which has a better leaving benzoyloxy group at C-3 as compared to the other two epoxides, should be most reactive. This is in agreement with the higher activity observed for (—)-pipoxide (IC50 = 8.35 tg/ml) as compared to the other two epoxides (IC50>

6

10

12

Acknowledgements This research was supported by grants from the University of Dar es Salaam, NUFFIC and NORAD. The London School of Hygiene and Tropical Medicine is acknowledged for providing the antimalarial testing facilities. We are grateful to Prof. H.

13 14 15

17

19

°

Achenbach (University of Erlangen, Germany), Prof. Dr. B. Zwanenburg (University of Nijmegen, The Netherlands), Dr. J. Wijnberg and Dr. M. Posthumus (University of Wageningen, The Netherlands) for providing spectra and for their helpful suggestions and Prof. J. D. Phillipson (London School of Pharmacy) for his useful comments during the preparation of the manuscript.

371. O'Neill, M. J., Bray, D. H., Boardman, P., Phillipson, J. D., Warhurst, D. c. (1985) Planta Med. 394. O'Neill, M. J., Bray, D. H., Boardman, P. P., Phillipson, J. D., Warhurst, D. c., Peters, W., Suffness, M. (1986) Antimicrob. Agents

chemother. 30, 101. chan, K. L., O'Neill, M. J., Phillipson, J. D., Warhurst, D. c. (1986)

25 g/ml). We would like to emphasize that the compounds isolated in this study were the major constituents. We are presently investigating whether more active minor constituents can be isolated from the plants listed in Table 1.

and training in Tropical diseases, Malaria, TDR 7th programme report, 1/1/83—31/12/84. WHO, Geneva, pp.2/1—2/67. World Health Organization (1959) Malaria eradication throughoul the world, WHO chronicle 13, 348.

22

Planta Med. 105. Bray, D. H., O'Neill, M. J., Phillipson, J. D., Warhurst, D.C. (1987) J. Pharmac. Pharmacol. 39, (suppl) 85. WHO (1984) Advances in malaria chemotherapy, Technical Report Series 711, 142. Leboeuf, M., cave, A., Bhaumik, P. K., Mukherjee, B., Mukherjee, R. (1982) Phytochemistry 21, 2783. Desjardins, H.P., Canfield, c. J., Hynes, J. D., chulay, J. D. (1979) Antimicrob. Agents chemother 16, 710. cole, J. R., Torrance, S. J., Wiedhopf, H. M., Arora, S. K., Bates, R. B. (1976)J. Org. chem. 41, 1852. Nkunya, M. H. H., Achenbach, H., Renner, c., Weenen, H. (1990) Phytochemistry, in preparation. Hufford, c. D., Oguntimein, B. 0. (1980) Phytochemistry 19, 2036. Lasswell, W. L., Jr., Huflord, c. D. (1976) Lloydia 39, 470. Tammami, B., Torrance, S. J., Fabela, F. V., Wiedhopf, H. M., Cole, J. R. (1977) Phytochemistry 16, 2040. Nkunya, M. H. H. (1985) J. Nat. Prod. 45, 999. Weenen, H., Nkunya, M. H. H., El-Fadi, A. A., Harkema, S., Zwanenburg, B. (1990)J. Org. chem. 55, 5107. Weenen, H., Nkunya, M. H. H., Mgani, Q. A., Achenhach, H., Posthumus, M. A. (1990) Phytochemistry, submitted. Nkunya, M. H. H., Weenen, H., Koyi, N.J. (1987) Phytochemistry 26, 2402. Nkunya. M. H. H., Weenen, H. (1989) Phytochemistry 28, 2217. Nkunya, M. H. H., Weenen, H., Koyl, N. J., Thijs, L., Zwanenburg, B. (1987) Phytochemistry 26, 2563.

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6'/C-7' double bond, as the 6',7'-dihydro isomer 11 is virtually inactive (1C50 = 50 .tg!ml). The high activity of 12 as compared to 10 is apparently dependent on the presence of the aj3-unsaturated hydroxyl group at C-8', which is absent in both 10 and 11.

Planta Med. 57(1991) 343

Antimalarial activity of Tanzanian plants and their active constituents: the genus Uvaria.

Petroleum ether, dichloromethane, and methanol extracts of leaves, stem, and root bark of nine Uvaria species: U. dependens, U. faulknerae, U. kirkii,...
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