Antiamoebic Activity of Iridoids from Morinda morindoides Leaves

• Abstract
• Materials and Methods
• References

Abstract

An aqueous decoction (dried extract), an 80 % methanolic extract from Morinda morindoides (Rubiaceae) leaves, and five iridoids isolated from the 80 % methanolic extract were evaluated in vitro for their activity against Entamoeba histolytica and their cytotoxicity. The aqueous decoction and the 80 % methanolic extract exhibited a promising antiamoebic activity with IC₅₀ values of 3.1 ± 1.7 and 1.7 ± 0.6 μg/mL, respectively. All tested iridoids displayed antiamoebic activity, the most active being epoxygaertneroside (IC₅₀: 1.3 ± 0.4 μg/mL) and methoxygaertneroside (IC₅₀: 2.3 ± 0.7 μg/mL) followed by gaertneroside, acetylgaertneroside and gaertneric acid with IC₅₀ values of 4.3 ± 1.8, 5.4 ± 1.4 and 7.1 ± 1.4 μg/mL, respectively. Synergistic effects between the iridoids tested, or with other constituents, may explain the high activity of the extracts. All extracts and iridoids were devoid of any cytotoxic effect against MT-4 cells at the highest test concentration of 250 μg/mL. These findings support at least in part the traditional use of Morinda morindoides leaves for the treatment of amoebiasis in the Democratic Republic of Congo.

Amoebic dysentery caused by Entamoeba histolytica is common in developing countries. Metronidazole is known to be a highly effective amoebicide and is considered to be the drug of choice for the treatment of amoebiasis, but due to its adverse effects and the emergence of drug resistance there is a need for new antiamoebic drugs [1], [2]. In developing countries many people rely on traditional medicine to treat this disease. Scientific validation and in vitro and/or in vivo evaluation of these traditional remedies are needed to prove their claimed effectiveness against amoebiasis. Therefore in the Democratic Republic of Congo, a research programme was initiated for the evaluation of traditional preparations from some medicinal plants used for the treatment of parasitic diseases such as amoebiasis [3] and malaria [4]. An aqueous decoction of the leaves of Morinda morindoides (Baker) Milne-Redhead (Rubiaceae), commonly called Nkongabululu (in Tshiluba) or Nkongobololo (in Lingala or Kikongo), which is used for the treatment of malaria, intestinal worms and amoebiasis, showed a promising antiamoebic activity. The present work deals with the evaluation of the in vitro antiamoebic activity of some extracts and isolated compounds from the leaves of this plant.

The aqueous decoction, which is the typical traditional preparation, and the 80 % methanolic extract from the leaves of M. morindoides exhibited antiamoebic activity with IC₅₀ values less than 5 μg/mL (Table [1]). The chloroform-, ethyl acetate-, n-butanol- and residual water-soluble fractions from the partitioning of the 80 % methanolic extract were evaluated as well, and the ethyl acetate- and n-butanol-soluble fractions showed IC₅₀ values of 3.1 ± 0.4 μg/mL and 1.5 ± 0.6 μg/mL, respectively. From these fractions a series of eight iridoid glycosides was isolated and identified as reported before, five of which were selected for the present biological testing, i. e., gaertneroside (1), acetylgaertneroside (2), gaertneric acid (3), methoxygaertneroside (4) and epoxygaertneroside (5) (Fig. [1]) [5]. The highest yield of iridoids, about 2.6 % (dry weight) was obtained from the n-butanolic fraction, which was also the most active one. The five tested iridoids showed a high antiamoebic activity with IC₅₀ values less than 10 μg/mL. The most active iridoids were epoxygaertneroside (5) and methoxygaertneroside (4) with IC₅₀ values of 1.3 ± 0.4 μg/mL and 2.3 ± 0.7 μg/mL, respectively. These results indicated that the presence of a methoxy group at C-3′ or an epoxy group between C-6 and C-7 was important for a pronounced antiamoebic activity. The presence of a carboxyl group at C-14 decreased the activity. Since the IC₅₀ values of the isolated compounds had the same order of magnitude as the crude extract and its active fractions, the accumulated effects of these iridoids could only account for part of the antiamoebic activity observed. Mutual synergistic effects of the iridoids, or with other compounds present in the active fractions such as flavonoid O-glycosides [6], may explain the high activity of the extracts and fractions.

This is the first report of the antiamoebic activity of this type of iridoids. Oruwacin, a ferulate iridoid isolated from Morinda lucida Benth. possessed a strong molluscicidal activity with LC₅₀ value < 10 ppm [7]. Iridoids isolated from Scrophularia lepidota Bois (Scrophulariaceae) exhibited an appreciable activity against the amastigote forms of Leishmania donovani and Trypanosoma brucei rhodesiense with IC₅₀ values ranging from 7 to 40 μg/mL, and some of them possessed a moderate antiplasmodial activity against a chloroquine- and pyrimethamine-resistant Plasmodium falciparum (K1) strain with no cytotoxic effect against an L-6 cell line [8].

Various plant families have been investigated in the search for antiamoebic constituents against Entamoeba histolytica in in vitro experiments [9], [10] and were found to be active. A wide range of phytochemicals such as alkaloids [9], [11], [12], [13], [14], [15], [16], quassinoids [9], [17], sesquiterpene lactones [18], [19], and flavonoids [20] were also evaluated, some of them demonstrating a high potency. The most active compounds were alkaloids isolated from some Strychnos spp., and quassinoids from Brucea spp. and members of the Simaroubaceae family, showing IC₅₀ values of 0.1 μg/mL. The antiamoebic activity of quassinoids was not correlated with their cytotoxicity [17]. Also for alkaloids it was reported that the antiprotozoal and cytotoxic effects did not necessarily parallel each other [13].

The cytotoxic activity of extracts, fractions and isolated compounds was evaluated against MT-4 cells during an anti-HIV screening in which they were found to be inactive. All test samples did not show any cytotoxic effect at the highest test concentration of 100 μg/mL. Therefore, the observed antiamoebic activity was not related to a non-specific cytotoxicity.

In conclusion, the aqueous decoction, some extracts and some iridoids present in the active fractions showed a pronounced antiamoebic activity. Synergistic effects between the iridoids tested, or with other constituents, may explain the high activity of the extracts. These findings support at least in part the traditional use of a decoction of Morinda morindoides leaves for the treatment of amoebiasis in the Democratic Republic of Congo.

Materials and Methods

Leaves of Morinda morindoides (Baker) Milne-Redhead (Rubiaceae) were collected in Kinshasa (DR Congo) in October 1989. The plant was identified by M. N. Nlandu of the Institut National d’Etudes et de Recherches en Agronomie (INERA) of the University of Kinshasa where a voucher specimen (INERA 891 055) has been deposited. The leaves were dried at room temperature and reduced to powder.

An aqueous decoction was prepared by boiling 20 g of fresh leaves for 15 min. The suspension was filtered and the resulting filtrate evaporated yielding a dried extract (5.62 g, 23.10 %). In the other experiment, dried and powdered leaves (500 g) were defatted by Soxhlet extraction with n-hexane. The dried plant material was macerated and percolated exhaustively with 80 % MeOH. The macerate and percolate were combined and evaporated to dryness under reduced pressure to give a dried extract (123.13 g, 24.63 %), 50 g of which were dissolved in distilled water (300 mL) and filtered. The filtrate was successively and exhaustively extracted with CHCl₃, EtOAc and n-BuOH. Each organic phase was evaporated to dryness to give the corresponding dried extracts: 4.43 g (8.4 %), 3.63 g (7.26 %) and 13.14 g (26.28 %), respectively. The remaining aqueous phase was also evaporated yielding 27.46 g of a dried extract (54.92 %). Iridoids named gaertenoroside (1), acetylgaertenoroside (2), gaertenic acid (3), methoxygaertenoroside (4) and epoxygaretenoroside (5) were isolated by column chromatographic methods and HPLC, and identified by conventional spectroscopic methods as described in a previous report [5].

The Entamoeba histolytica strain used in this investigation was a laboratory strain isolated from patients with acute amoebic dysentery, and kindly provided by the Institute of Tropical Medicine, Faculty of Medicine, University of Kinshasa. The maintenance of Entamoeba histolytica cultures on Dobbell and Laidlaw’s medium as well as the evaluation of the antiamoebic activity of plant extracts, fractions and isolated pure compounds were performed according to the procedure previously described [3]. Tests were carried out in triplicate. Results are expressed as IC₅₀ values. Metronidazole (Medeva Pharma; Braine-l’Alleud, Belgium) was used as a reference antiamoebic product.

The cytotoxicity of extracts, fractions and isolated compounds was tested against MT-4 cells during an anti-HIV screening [21]. All samples were tested in triplicate, and CC₅₀ values calculated.

Student’s t-test was used to determine p values for the differences observed between the test samples and between the test samples and the controls. A p value of 0.05 or less was considered indicative of a statistically significant difference.

References

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  CrossrefPubMedSearch in Google Scholar

Kanyanga Cimanga

Department of Pharmaceutical Sciences

University of Antwerp

Universiteitsplein 1

2610 Antwerp

Belgium

Phone: +32-3-820-2647

Fax: +32-3-820-2709

Email: kanyanga.cimanga@ua.ac.be

References

• 1 Orozco E, Pérez D G, Gómez M C, Ayala P. Multidrug resistance in Entamoeba histolytica .  Parasitol Today. 1995;  11 473-5
  CrossrefPubMedSearch in Google Scholar
• 2 Sharma P, Sharma J D. A review of plant species assessed in vitro for antiamoebic activity or both antiamoebic and antiplasmodial properties.  Phytother Res. 2001;  15 1-17
  CrossrefPubMedSearch in Google Scholar
• 3 Tona L, Kambu K, Ngimbi N, Cimanga K, Vlietinck A J. Antiamoebic and phytochemical screening of some Congolese medicinal plants.  J Ethnopharmacol. 1998;  61 57-65
  CrossrefPubMedSearch in Google Scholar
• 4 Tona L, Ngimbi N P, Tsakala M, Mesia K, Cimanga K, Apers S. et al . Antimalarial activity of 20 crude extracts from nine African medicinal plants used in Kinshasa, Congo.  J Ethnopharmacol. 1999;  68 193-203
  CrossrefPubMedSearch in Google Scholar
• 5 Cimanga K, Hermans N, Apers S, Van Miert S, Van den Heuvel H, Claeys M. et al . Complement-inhibiting iridoids from Morinda morindoides .  J Nat Prod. 2003;  66 97-102
  CrossrefPubMedSearch in Google Scholar
• 6 Cimanga K, De Bruyne T, Lasure A, Qimin L, Pieters L, Claeys M. et al . Flavonoid O-glycosides from the leaves of Morinda morindoides .  Phytochemistry. 1995;  38 1301-3
  CrossrefPubMedSearch in Google Scholar
• 7 Adewunmi C O, Adesogan E K. Antraquinones and oruwacin from Morinda lucida as possible agents in fascioliasis and schistosomiasis control.  Fitoterapia. 1984;  57 259-63
  PubMedSearch in Google Scholar
• 8 Tasdemir D, Güner N D, Perozzo R, Brun R, Dönmez A A, Çalis I. et al . Anti-protozoal and plasmodial FabI enzyme inhibiting metabolites of Scrophularia lepidota root.  Phytochemistry. 2005;  86 355-62
  PubMedSearch in Google Scholar
• 9 Keene A T, Harris A, Phillipson J D, Warhurst D C. In vitro amoebicidal testing of natural products. Part I. Methodology.  J Nat Prod. 1986;  52 278-85
  PubMedSearch in Google Scholar
• 10 Van Beek T A, Deelder A M, Verpoorte R, Svendsen A B. Antimicrobial, antiamoebic and antiviral screening of some Tabermaemontana sp.  Planta Med. 1984;  50 180-5
  PubMedSearch in Google Scholar
• 11 Keene A T, Phillipson J D, Warhurst D C, Koch M, Seguin E. In vitro amoebicidal testing of natural products. Part 2. Alkaloids related to emetine.  Planta Med. 1987;  53 201-6
  Thieme ConnectPubMedSearch in Google Scholar
• 12 Bhutani K K, Sharma G L, Ali M. Plant based antiamoebic drugs. Part I. Antiamoebic activity of phenanthroindolizidine alkaloids; common structural determinants of activity with emetine.  Planta Med. 1987;  53 532-6
  PubMedSearch in Google Scholar
• 13 Wright C W, Bray D H, O’Neill M J, Warhurst D C, Phillipson J D, Quetin-Leclercq J. et al . Antiamoebic and antiplasmodial activities of alkaloids isolated from Strychnos usambarensis .  Planta Med. 1991;  57 337-40
  PubMedSearch in Google Scholar
• 14 Wright C W, Allen D, Cai Y, Phillipson J D, Said I M, Kirby G C. et al . In vitro antiamoebic and antiplasmodial activities of alkaloids from Alstonia angustifolia roots.  Phytother Res. 1992;  6 121-4
  PubMedSearch in Google Scholar
• 15 Wright C W, Allen D, Chen Z, Phillipson J D, Kirby G C, Warhurst D C. et al . Selective antiprotozoal activity of some Strychnos alkaloids.  Phytother Res. 1994;  8 149-52
  PubMedSearch in Google Scholar
• 16 Marshall S J, Russel P F, Wright C W, Anderson M M, Phillipson J D, Kirby G C. et al . In vitro antiplasmodial, antiamoebic and cytotoxic activities of a series of benzylisoquinoline alkaloids.  Antimicrob Agents Chemother. 1994;  36 96-103
  PubMedSearch in Google Scholar
• 17 Wright C W, O’Neill M J, Phillipson J D, Warhurst C D. Use of microdilution to assess in vitro antiamoebic activities of Brucea javanica fruits, Simarouba amara stem, and a number of quassinoids.  Antimicrob Agents Chemother. 1988;  32 1725-9
  CrossrefPubMedSearch in Google Scholar
• 18 Sharma P K, Bhutani K. Plant based antiamoebic drugs. Part II. Amoebicidal activity of parthenin isolated from Parthenium hysterophorus .  Planta Med. 1988;  54 120-2
  PubMedSearch in Google Scholar
• 19 Yu H W, Wright C W, Cai Y, Yang S L, Phillipson J D, Kirby G C. et al . Antiprotozaol activities of Centipeda minima .  Phytother Res. 1994;  8 436-8
  PubMedSearch in Google Scholar
• 20 Calzada F, Meckes M, Cedillo-Rivera R. Antiamoebic and antigiardial activity of plant flavonoids.  Planta Med. 1999;  65 78-80
  Thieme ConnectPubMedSearch in Google Scholar
• 21 Pauwels R, Balzarini J, Baba M, Snoeck R, Schols D, Herdewijn P. et al . Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds.  J Virol Methods. 1988;  20 309-21
  CrossrefPubMedSearch in Google Scholar

Kanyanga Cimanga

Department of Pharmaceutical Sciences

University of Antwerp

Universiteitsplein 1

2610 Antwerp

Belgium

Phone: +32-3-820-2647

Fax: +32-3-820-2709

Email: kanyanga.cimanga@ua.ac.be