In vitro antiplasmodial activity of extracts and fractions from seven medicinal plants used in the Democratic Republic of Congo

Journal of Ethnopharmacology 93 (2004) 27–32

In vitro antiplasmodial activity of extracts and fractions from seven medicinal plants used in the Democratic Republic of Congo L. Tona a,b , R.K. Cimanga c,∗ , K. Mesia a , C.T. Musuamba a , T. De Bruyne c , S. Apers c , N. Hernans c , S. Van Miert c , L. Pieters c , J. Totté c , A.J. Vlietinck c b

a Faculty of Pharmacy, University of Kinshasa, B.P. 212, Kinshasa XI, Democratic Republic of the Congo Institute of Tropical Medicine, Faculty of Medicine, University of Kinshasa, B.P. 834, Kinshasa XI, Democratic Republic of the Congo c Department of Pharmaceutical Sciences, University of Antwerp (UA), Universiteitsplein 1, B-261O, Antwerp, Belgium

Received 11 February 2004; received in revised form 23 February 2004; accepted 25 February 2004 Available online 18 May 2004

Abstract The in vitro antiplasmodial activity of seven EtOH extracts and twenty fractions from the partition of the initial ethanolic extracts from seven African medicinal plants used in the Democratic Republic of Congo (DR Congo) for the treatment of malaria was evaluated. The most active EtOH extracts (IC50 < 3 ␮g/ml) were those from Cassia occidentalis leaves, Euphorbia hirta whole plant, Garcinia kola stem bark and Phyllanthus niruri whole plant. Their respective petroleum ether soluble fractions also exhibited an antiplasmodial activity with IC50 < 3 ␮g/ml. EtOH extracts from Vernonia amygdalina leaves (5 < IC50 < 10 ␮g/ml), Tetracera poggei leaves (10 < IC50 < 50 ␮g/ml) and Morinda morindoides leaves (50 < IC50 < 100 ␮g/ml) were less active, but their petroleum ether fractions exhibited a pronounced antiplasmodial activity (IC50 < 3 ␮g/ml). The same observation could also be made for the petroleum ether fraction from Cassia occidentalis, Euphorbia hirta, Garcinia kola and Phyllanthus niruri. Isoamyl alcohol fractions from Euphorbia hirta, Phyllanthus niruri and Vernonia amygdalina showed IC50 values less than 3 ␮g/ml, and from Cassia occidentalis, Garcinia kola, Morinda morindoides and Tetracera poggei between 10 and 50 ␮g/ml. The observed antiplasmodial activity may be related to the presence of terpenes, steroids, coumarins, flavonoids, phenolic acids, lignans, xanthones and anthraquinones. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Cassia occidentalis; Euphorbia hirta; Garcinia kola; Morinda morindoides; Phyllanthus niruri; Tetracera poggei; Vernonia amygdalina; Extracts; Fractions; Malaria; In vitro antiplasmodial activity

1. Introduction Human malaria is one of the most important health problems in tropical and subtropical regions. The widespread resistance of Plasmodium falciparum against classical antimalarial drugs through the tropics (Olliaro and Yuthavong, 1999) has led to a search of new drugs with new modes of action. The search for new remedies from medicinal plant species used for the treatment of malaria depends on the accurate and specific ethnobotanical and ethnopharmacological information obtained from local healers. In the Democratic Republic of Congo, a research programme was initiated for the evaluation of the antiamoebic (Tona et al., 1999a) and antiplasmodial activity (Tona et al., 1999b) of medicinal plant species used for the treatment of ∗ Corresponding

author. Tel.: +32-3820-2742; fax: +32-3820-2709. E-mail address: [email protected] (R.K. Cimanga).

amoebiasis and malaria in Congolese traditional medicine. The present study deals with the partition of the total EtOH extracts from some plant species which have shown an interesting antimalarial activity in our previous investigation, that is, Cassia occidentalis, Euphorbia hirta, Garcinia kola, Morinda morindoides, Phyllanthus niruri, Tetracera poggei and Vernonia amygdalina, with the aim to locate active fractions. 2. Materials and methods 2.1. Experimental section TLC was carried out on Silica gel 60 F254 Merck (layer thickness 0.25 mm). Betunilic acid, ellagic acid and lupeol were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Quercetin was obtained from Carl Roth GmbH, Germany. 1,8-Dihydroxyanthraquinone was isolated from

0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.02.022

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L. Tona et al. / Journal of Ethnopharmacology 93 (2004) 27–32

Table 1 Antimalarial plant species selected Plant names and family

Voucher specimen no.

Plant parta

Dry weight of extracts (%)b A

B

C

D

E

Cassia occidentalisL. (Caesalpiniaceae) Euphorbia hirta L. (Euphorbiaceae) Garcinia kola Heckel (Clusiaceae) Morinda morindoides (Baker) Milne-Redhead (Rubiaceae) Phyllanthus niruri L. (Euphorbiaceae) Tetracera poggei Gilg. Dilleniaceae) Vernonia amygdalina Del. (Asteraceae)

P10897 NL P11O89 NL P120897 NL P020897 NL

Leaf Whole plant Stem bark Leaf

5.3 3.8 15.8 6.0

1.4 1.4 2.4 4.3

2.4 1.2 3.3 2.1

3.2

1.5

4.1 1.5

1.2

P090897 NL P250897 NL P150897 NL

Whole plant Leaf Leaf

12.0 15.8 6.2

2.1 1.1 1.7

5.2 6.2 2.7

2.5

a A: ethanolic extract; B: petroleum ether extract; C and D: isoamyl alcohol and chloroformic fractions, respectively, obtained at pH 2–3; E: chloroformic fraction obtained at pH 10. b %: from dry plant material.

Morinda morindoides leaves. All chemicals were of highest purity. 2.2. Plant materials All plants (Table 1) were collected in Kinshasa, Democratic Republic of Congo (DR Congo) in August 1997. They were identified by Mr. N. Nlandu of the Institut National d’Etudes et de Recherches en Agronomie (INERA) of the University of Kinshasa by comparison with voucher specimen references. A voucher specimen for each plant has been deposited in the herbarium of this Institute. 2.3. Preparation of extracts Dried and powdered plant material (50 g) of each plant part was maecrated with 300 ml EtOH for 24 h (3×), filtered and the filtrate evaporated to dryness in vacuo yielding dried extracts denoted as extract A. Five grams of each ethanolic extract was separately dissolved in 50 ml distilled water and exhaustively extracted with petroleum ether which was evaporated in vacuo to give semi-dried extracts denoted as extract B. The remaining aqueous phase was acidified (HCl 2 N, pH 2–3), boiled for 30 min and successively extracted with isoamyl alcohol and CHCl3 . Each organic phase was evaporated in vacuo to give corresponding dried extracts C and D, respectively. On the other hand, 5 g of extract A from Cassia occidentalis leaves, Morinda morindoides leaves and Phyllanthus niruri whole plant was separately dissolved in 50 ml distilled H2 O and filtered, then the filtrate was alkalinised with NH4 OH 10% (pH 10). The mixture was exhaustively extracted with CHCl3 which was evaporated to dryness (extract E) (Harborne, 1973). 2.4. TLC analysis of extracts and fractions Two milligrams of each extract and fraction was separately dissolved in 2 ml MeOH. The identification of major chemical groups was carried out by TLC on silica gel 60 F254 Merck (layer thickness 0.25 mm) as follows; for polyphenols (flavonoids), n-butanol/acetic acid/water

4:1:5 (top layer) was used as solvent system, 1% FeCl3 and 5% AlCl3 (MeOH solution) as reagents. Alkaloids were detected with Dragendorff’s reagent and ethylacetate/isopropanol/ammonia: 16:3:1 was used as mobile phase. Anthraquinones were revealed with NaOH 10% or ammonia 25% using ethylacetate/methanol/water: 8:1:1 as mobile phase. Coumarins were detected under UV (366 nm) thanks to their blue fluorescence which becomes intense after spraying KOH 10%. Terpenes or steroids were identified with Liebermann–Burchard, as reagent using n-hexane/CH2 Cl2 : 1:9 as mobile phase. Aqueous solutions were used to detect saponins by the froth test (Harborne, 1973). 2.5. In vitro test for antiplasmodial activity The in vitro antiplasmodial activity was evaluated according to the method described by Tona et al. (1999b). Five milligrams of each test sample was dissolved in 5 ml EtOH and diluted with culture medium to give a series of test concentrations ranging from 0.5 to 500 ␮g/ml which were tested in triplicate against Plasmodium falciparum chloroquine sensitive infected human blood. Quinine 2HCl from Laboratoire d’Analyse et de Contrˆole des Médicaments et des Denrées Alimentaires (LACOMEDA), Faculty of Pharmacy, University of Kinshasa, was used as an antimalarial reference product. IC50 values were derived from dose–response curves. 2.6. Statistical analysis The Student’s t-test was used to test the significance of the difference between results obtained for different samples, and between results for samples and controls. Statistical significance was set at P = 0.05

3. Results and discussion The results from the in vitro antiplasmodial testing of 7 EtOH extracts and 20 fractions from the partition of their

L. Tona et al. / Journal of Ethnopharmacology 93 (2004) 27–32 Table 2 In vitro antiplasmodial activity of some plant extracts and their fractions Botanical names

Plant part

Extracts/ fractionsa

IC50 (␮g/ml)b

Cassia occidentalis

Leaf

A B C D E

2.8 1.5 18.6 2.9 >1000

Euphorbia hirta

Whole plant

A B C

Garcinia kola

Stem bark

A B C D

2.9 1.6 41.7 27.1

± ± ± ±

0.7 0.2 3.2 2.7

Morinda morindoides

Leaf

A B C D E

94.2 1.8 15.3 8.8 >1000

± ± ± ±

3.4 0.2 3.6 2.5

Phyllanthus niruri

Whole plant

A B C E

2.5 ± 0.1 1.3 ± 0.3 2.3 ± 0.5 >1000

Tefracera pogge

Leaf

A B C

36.9 ± 4.2 1.7 ± 0.4 21.8 ± 5.2

Vernonia amygdalina

Leaf

A B C

9.7 ± 2.6 2.5 ± 0.7 2.7 ± 0.6

Quinine 2HCl a b

± ± ± ±

0.5 0.7 3.6 0.3

2.4 ± 0.2 1.2 ± 0.3 2.6 ± 1.2

0.25 ± 0.02

See Table 1. n = 3.

respective initial EtOH extracts are presented in Table 2. With regard to the ethanolic extract A, a high antiplasmodial activity (IC50 < 3 ␮g/ml) was observed for Cassia occidentalis leaves, Euphorbia hirta, Garcinia kola and Phyllanthus niruri, whereas, Vernonia amygdalina and especially Tetracera poggei and Morinda morindoides were less active. However, the petroleum ether soluble fractions of the total extract of all plants tested were highly active (IC50 < 3 ␮g/ml), indicating the presence of lipophilic active constituents in all plants. For only three plants (Euphorbia hirta, Phyllanthus niruri and Vernonia amygdalina), the isoamyl alcohol soluble fraction (C) showed a comparably (P > 0.05) high activity while that from Cassia occidentalis, Garcinia kola and Tetracera poggei showed a lower activity (P < 0.001) than fractions A and B from the same plant material. The same observation could be made for the chloroform soluble fraction prepared at pH 2–3 (D) of Cassia occidentalis. The chloroform soluble fractions prepared at pH 10 (E) were not active. Cassia occidentalis L. (Caesalpiniaceae) is widely used in traditional medicine in some African countries for the treatment of various diseases according to the used plant part

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(Tona et al., 1999b, 2001). In the present investigation, the EtOH extract A from the leaves, its petroleum ether fraction B and CHCl3 fraction D obtained at pH 2–3 showed a high in vitro antiplasmodial activity (IC50 < 3 ␮g/ml). No significant difference in their activity was statistically observed (P > 0.05). The aqueous decoction of the leaves has been shown to produce 55% inhibition of Plasmodium falciparum in vitro (Gbeassor et al., 1988), the lyophilisated extract from the decoction of the leaves has been reported to inhibit Plasmodium falciparum FCC2 chloroquine sensitive from Niger and F2R chloroquine resistant strain from Comores with IC50 values between 500 and 700 ␮g/ml (Gasquet et al., 1993). In addition, Ali et al. (2002) have also shown that a 80% MeOH extract from Cassia occidentalis leaves exhibited an antiplasmodial activity against K1 chloroquine resistant and NF54 chloroquine sensitive Plasmodium falciparum with IC50 of 4.96 and 2.21 ␮g/ml, respectively. TLC analysis of the Cassia occidentalis samples described here revealed the presence of terpenes or steroids (fraction B), flavonoids (fraction C) and anthraquinones and derivatives (fraction D) as well as in the extract A, as also reported in the literature, especially that of 1,8-dihydroxyanthraquinone (Kerharo and Adam, 1974; Oliver-Bever, 1986; Kambu, 1990). This anthraquinone could be considered as the one of the active constituents because of its in vitro antimalarial effect already reported (Cimanga, 1997). On the other hand, the activity observed in the petroleum ether, isoamyl alcohol and CHCl3 (pH 2–3) fractions could be related to the presence of terpenes and/or steroids, flavonoids and anthraquinones. Representatives of these classes have already been reported to inhibit Plasmodium falciparum growth in vitro and/or in vivo (Wright and Phillipson, 1990; Christensen and Kharazmi, 2001). Euphorbia hirta L. (Euphorbiaceae) whole plant is used in tropical and subtropical countries for the treatment of various diseases such as asthma, diarrhea and amoebiasis (Tona et al., 1999a; Kambu, 1990). In our present antimalarial screening, the EtOH extract A from the whole plant, its petroleum ether fraction B and isoamyl alcohol fraction C exhibited a high antiplasmodial activity (IC50 < 3 ␮g/ml). No significant difference in the activity of these Euphorbia hirta samples was statistically (P > 0.05) observed. Phytochemical screening revealed the presence of terpenes or steroids, flavonoids as aglycones (methoxylated flavonoids) in fraction B and flavonoids in fraction C as well as saponins in the extract A. Terpenes (euphorbol derivatives), saponins, tannins, anthocyanins and flavonoids among which methoxylated flavonoids such as 3,3 -dimethoxy-4 ,5,7-trihydroxyflavone and 3,4 -dimethoxy-3,5,7-trihydroxyflavone, quercetin and derivatives (Bakana, 1984), phenolic acids such as ellagic acid (Oliver-Bever, 1986) have been reported before. Quercetin and ellagic acid can be considered as some of the active constituents of this plant because of their antiplasmodial activity earlier reported (Cimanga, 1997; Banzouzi et al., 2002). Furthermore, methoxylated flavonoids,

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terpenes and steroids (Phillipson and Wright, 1991; Kraft et al., 2003) and saponins (Oketch-Raban and Dossaji, 1997; Traore et al., 2000) have been reported to exhibit an antiplasmodial activity. The stem bark Garcinia kola Heckel (Clusiaceae) is used in some African regions as an aqueous or a palm wine macerate to treat diarrhea, but it is not used against malaria or fever while the seed is reputed for its aphrodisiac properties and for the treatment of malaria (Kerharo and Adam, 1974; Kambu, 1990). The stem bark was selected in our antiplasmodial screening on the basis of information obtained from a traditional healer. The results from the present antiplasmodial screening indicated that the EtOH extract A and its petroleum ether fraction B showed IC50 values less than 3 ␮g/ml suggesting a similar activity (P > 0.05). Phenolic acids, tannins, triterpenoids, sterols, polyisoprenylated benzophenones, flavonoids and biflavonoids (Kerharo and Adam, 1974; Bakana, 1984) have been reported to be present in Garcinia kola stem bark. Other species belonging to this genus are known to contain in addition xanthones (Bakana, 1984). During our study, TLC analysis of the EtOH extract stem bark, its petroleum ether and isoamyl alcohol fractions revealed the presence of terpenes or steroids, xanthones and flavonoids. Xanthones isolated from other Garcinia species have been shown to have an in vitro antimalarial activity (Likhiwitayawid et al., 1998a,b). Therefore, the possibility that the antiplasmodial activity displayed by Garcinia kola samples reported here would be due to the presence of these kind of compounds could not be excluded. An aqueous decoction from the leaves of Morinda morindoides (Baker) Milne-Redhead (Rubiaceae) is used in some African countries for the treatment of some diseases such as fever including malaria, amoebiasis, haemorrhoids, intestinal worms and gonorrhea, as an anti-rheumatic remedy and tonic (Kambu, 1990). In relation to its use for the treatment of rheumatic pains, a series of complement-inhibiting flavonoids (Cimanga et al., 1995; Cimanga, 1997) and iridoids (Cimanga et al., 2003) have been isolated from the leaves. Flavonoids have been also shown to act as inhibitors of xanthine oxidase activity and scavengers of superoxide anion (Cimanga et al., 1999). A 80% MeOH extract from the leaves has been shown to be inactive against Plasmodium falciparum NF54, a chloroquine sensitive strain (IC50 > 100 ␮g/ml). Alizarin and chrysarin (10 < IC50 < 25 ␮g/ml) isolated from the chloroformic fraction from the partition of the 80% MeOH extract as well as quercetin (IC50 < 6 ␮g/ml) from the initial extract have been shown to exhibit an antiplasmodial activity against the same parasite (Cimanga, 1997). In the present antiplasmodial screening, it was observed that the petroleum ether fraction (B) was the most active one (1 < IC50 < 2 ␮g/ml). In addition to known antiplasmodial constituents cited above, terpenic and steroidal compounds detected by TLC analysis during the present work could also account for the activity of the extract and fractions for the same reasons mentioned above.

Phyllanthus niruri L. (Euphorbiaceae) whole plant is used in some African countries for the treatment of dysentery, malaria and intestinal spasms, and for other indications (Kerharo and Adam, 1974; Oliver-Bever, 1986). In previous biological studies, an ethanolic extract from the whole plant has been reported to exhibit antibacterial, antifungal and antiviral activities (Calixto et al., 1998). It also displayed an antiamoebic effect (Oliver-Bever, 1986; Tona et al., 1999a), but a 50% EtOH extract from the same plant collected in India failed to inhibit the malaria parasite P. berghei berghei in vivo (Kerharo and Adam, 1974; Oliver-Bever, 1986). This finding is in disagreement with the in vivo antiplasmodial activity recently reported by Usha Devi et al. (2001) for the same extract. Its analgesic activity was also reported (Santos et al., 1995) and could be attributed to the presence of geranin which has been shown to be responsible for the same pharmacological activity detected in an ethanolic extract from Phyllanthus sellowianus (Miguel et al., 1996). The results from the present biological activity screening indicated that the EtOH extract A from the whole plant, its petroleum ether and isoamyl alcohol fractions showed a similar activity (P > 0.05) against Plasmodium falciparum with IC50 values of 2.5 ± 0.1, 1.3 ± 0.3 and 2.3 ± 0.5 ␮g/ml, respectively, while fraction E was inactive. Terpenic or steroidal compounds, and flavonoids were identified by TLC in fractions B and C, respectively, while alkaloids were unambiguously detected in fraction E. Previous phytochemical studies on this plant have reported the isolation and identification of flavonoids such as quercetin and derivatives, lignans, terpenoids such as lupeol and derivatives, steroids, phenolic acids such as ellagic acid, and alkaloids such as nirurine and derivatives (Calixto et al., 1998). Therefore, its seems that the antiplasmodial activity of Phyllanthus niruri extract and fractions would be related to the presence of ellagic acid, quercetin, lupeol and lignans for which the antiplasmodial activity was already described in both in vitro and/or in vivo (Wright and Phillipson, 1990; Alves et al., 1997; Cimanga, 1997; Christensen and Kharazmi, 2001; Banzouzi et al., 2002). An aqueous decoction from the leaves of Tetracera poggei Gug. (Dilleniaceae) is used in traditional medicine against dysentery, hepatitis, blennorrhagia, as febrifuge and diuretic agent (Tangenyi, 1996). Only rhamnocitrin 3-sulphate has been isolated from this species (Gurni et al., 1981), though some triterpenoids (Ma et al., 1999) and flavonoids (Harrison et al., 1994) have been isolated from other species such as Tetracera boiviana and Tetracera indica, respectively. In this work, a high antiplasmodial activity was found in the petroleum ether fraction B (IC50 < 2 ␮g/ml). TLC analysis of the EtOH extract and its fractions indicated the presence of saponins, terpenes such has betulinic acid, steroids and flavonoids as major constituents. Betulinic acid could be considered as the one of the antiplasmodial compounds for this plant because of its antimalarial properties already reported (Bringmann et al., 1997; Steele et al., 1999).

L. Tona et al. / Journal of Ethnopharmacology 93 (2004) 27–32

An aqueous decoction from the leaves of Vernonia amygdalina Delide (Asteraceae) is used for various purposes (Oliver-Bever, 1986; Kambu, 1990). The antiaggregating and disaggregating activity during rabbit platelet aggregation of an alcoholic extract from the fruits was attributed to vernolepin and its mechanism of action was also described (Laekeman et al., 1982, 1985). Chimpanzees are also known to chew the pith of the young shoots as a way of treating parasitic infections (Huffman and Seifu, 1989). The active constituents have been reported to be the sesquiterpene lactones vernodalin, vernolide and hydroxyvernolide, and steroid-related constituents vernonioside BI and vernoniod BI (Koshimizu et al., 1994). Antimicrobial (Akinpelu, 1999; Tawo et al., 1999; Kambizi and Afolayan, 2001) and cathartic activities (Awe and Makinde, 1999) have been reported and the isolation of the active constituents have been also described in some cases (Al Magboul et al., 1997). In the present investigation, especially fractions B and C showed a high activity (IC50 = 2.5 ± 0.7 and 2.7 ± 6 ␮g/ml, respectively). No significant difference in their activity was statistically (P > 0.05) observed. Our results are qualitatively consistent with those reported for the polar extracts by Masaba (2000). Other extracts from Vernonia species such as the n-hexane from leaves of Vernonia brasiliana (L) Druce showed an in vitro antiplasmodial activity which was attributed to lupeol (Alves et al., 1997). Sesquiterpene lactones such as vernolepin, vernolin, vernolide, vernodalin and hydroxyvernodalin isolated from Vernonia amygdalina leaves have been reported to exhibit an antiplasmodial activity (IC50 < 4 ␮g/ml) against Plasmodium falciparum strains (Phillipson et al., 1993). In the present work, TLC analysis revealed the presence of saponins, terpenes, steroids, coumarins and flavonoids. It cannot be excluded that, in addition to the sesquiterpene lactones, other antiplasmodial constituents are present in Vernonia amygdalina as well. For extracts of Cassia occidentalis leaves, Morinda morindoides leaves and Phyllanthus niruri whole plant, the in vivo antiplasmodial activity has already been reported (Tona et al., 2001). A good correlation between the in vitro and in vivo antiplasmodial activity of these selected medicinal plant species exists. The analgesic and antipyretic properties already reported for Euphorbia hirta (Langhers et al., 1991) and P. niruiri (Santos et al., 1995) can also help to reduce the fevers as a result of malaria attacks. All reported active constituents at least responsible for the antiplasmodial activity observed in extracts and fractions in the present investigation were unambiguously identified by TLC analysis in the presence of reference products. Our further works aim at the isolation and structure elucidation of more other active principles.

Acknowledgements This biological investigation is dedicated to the memory of Prof. Dr. P.N. Ngimbi of the Institute of Tropical Medicine,

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Faculty of Medicine, University of Kinshasa for its enthusiasm and its active scientific contribution to our project on the evaluation of the antimalarial activity of some medicinal plants used for the treatment of malaria in Congolese traditional medicine. All technicians of this Institute are acknowledged for their technical assistance.

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