|Iranian Journal of Pharmaceutical Research (2005) 2: 101-103
Received: June 2004
Accepted: February 2005
Copyright ? 2005 by School of Pharmacy
Soodabeh Saeidniaa*, Ahmad Reza Goharia, Fumiyuki Kiuchib and Gisho Hondac
aMedicinal Plants Research center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. bTsukuba Medicinal Plant Research Station, National Institute of Health Sciences, Ministry of Health, Labour and Welfare, Tsukuba, Ibaraki, Japan. cDepartment of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
* Corresponding author: firstname.lastname@example.org
Some medicinal plants are a potential source of new drugs, in order to improve the treatment of Chagas disease whose treatment is still a challenge. In this study, the in vitro anti-epimastigote activity of certain fractions of Achillea biebersteinii, A. millefolium, Satureja mutica and S. macrantha was evaluated. Diethyl ether fractions of Achillea species and acetone fractions of Satureja species were the most active fractions (MLC=12.5 μg/ml) against the epimastigotes of Trypanosoma cruzi, the ethiological agent causing Chagas disease. The trypanocidal activity seems to be decreased by fractionation, using MeOH and water as the solvents. The results obtained from assay revealed that Achillea and Satureja species could be a source of active trypanocidal compounds.
Chagas disease is caused by the flagellete protozoan Trypanosoma cruzi (Trypanosomatina), leading to approximately 400,000 deaths per year (1). Trypomastigotes ingested by the insect differentiates into the proliferative epimastigote form that, on reaching the posterior intestine, evolves to metacyclic trypomastigotes. The latter form undergoes differentiation into amastigotes, which after several reproductive cycles transform to trypomastigotes, the form responsible for the dissemination of the infection (2). Current treatment is unsatisfactory, because the only two available drugs, benznidazole and nifortimox, possess severe side effects and their activity is limited to the acute phase (3).
Recently, higher plants belonging to the Rutacea, Meliaceae, Simaroubaceae and Burceraceae families have been studied in an attempt to find active compounds against Trypanosoma cruzi, the causative agent of Chagas disease (1). The genus Achillea (Compositae) is well-known for medicinal properties such as anthelmintic, anti-inflammatory and anti- microbial effects (4, 5). Achillea millefolium L. and Achillea biebersteinii Afan. are two of among nineteen herbaceous species growing in the northen parts of Iran (6). The genus Satureja (Labiatae) has been used in traditional Medicine for its carminative, tonic, stomachique, diuretic and anti-cancer activities. They are also used for rheumatic pain and asthma (4). S. mutica Fisch et C. A. Mey and S. macrantha C. A. Mey are two of eight endemic species of Satureja growing in north of Iran (7). There is no paper to investigate the anti-trypanosoma effect of these species. In this study it was decided to examine their activity against the epimastigotes of T. cruzi.
Achillea millefolium and A. biebersteinii were collected from Kord Kooy at Golestan State of Iran in August 1999 (during full flowering stage) and identified by Dr. H. Akhani. The voucher herbarium specimens (No.13607 for A. millefolium and No. 13606 for A. biebersteinii) were deposited in the private herbarium of Dr. H. Akhani, housed at the Department of Biology, Faculty of sciences, Tehran University. Satureja mutica and S. macrantha were collected in September 2000 from Guilan and West Azarbayjan states in Iran, respectively, and identified by Dr. V. Mozaffarian. The voucher herbarium specimens (TARI- 78411 for S. mutica and TARI- 78409 for S. macrantha) were deposited at the herbarium of Research Institute of Forests and Rangelands.
Preparing the extracts
Aerial parts of the plants (flowers, leaves and stems) were dried carefully and reduced to powder, followed by extraction three times with diethyl ether via maceration at room temperature for 72 h. This process was repeated on the marc with ethyl acetate (for Achillea) or acetone (for Satureja), methanol and water, successively, and then the solvents evaporated under reduced pressure to obtain the concentrated extracts. All extracts were dried under vacuum in order to give dried powder extracts.
Evaluation of anti-epimastigote activity
Epimastigotes of T. cruzi (Tulahuen strain) were kept in GIT medium (Wako) supplemented with hemin (12.4 ?M, Wako). The epimastigotes in GIT medium (10 ?L) were incubated with a test sample (extracts) dissolved in EtOH (5 ?L) and autoclaved saline (185 ?L). All samples were incubated at 27˚C for 24 h. The movement of epimastigotes was observed under a microscope. It was assumed that immobilized organisms had been killed. The control contained ethanol in the same proportion utilized to dissolve the drugs. Each assay was performed in duplicate. Gentian violet was used as a positive control and its minimum lethal concentration was found to be 6.3 ?M (8-10).
Results and discussion
In the present study, the trypanocidal activity of 16 fractions (diethyl ether, acetone, methanol and water extracts) from Achillea and Satureja species was evaluated. Table 1 summarizes the results obtained from fractionation and assay and shows that the diethyl ether and ethyl acetate fractions of both Achillea plants were active against the epimastigote of T. cruzi. MeOH fraction of A. biebersteinii shows a better trypanocidal effect than A. millefolium. Among several fractions of Achillea, only aqueous extracts did not show any activity at concentrations used in this study. Table 1 shows that the diethyl ether and acetone fractions of both Satureja plants were active against T. cruzi. Diethyl ether fractions of S. mutica showed a better trypanocidal effect than S. macrantha. Trypanocidal activity seems to be enriched by fractionation, using acetone as a solvent, and all the epimastigotes were completely eliminated. Only methanol and aqueous fractions of the plants did not show any activity at concentrations used in this study. Detailed explanation of the observed activity of these fractions must await the results of the ongoing phytochemical studies and in vivo bioassay of the isolated compounds against the parasite.
In the concept of efforts to improve the therapy of Chagas disease, higher plants appear to be a potential source of new drugs, with high activity and low toxicity. A broad spectrum of chemical classes of substances could show activity against the parasite. (11). It is possible that the activity of the Labiatae family could be associated with the terpenoids and flavonoids (12, 13). Achillea is well-known as a source of methoxylated flavonoids, which could be the main cause of activity against the Trypanosoma cruzi (13, 14).
In conclusion, the Iranian species of Satureja and Achillea (especially non-polar fractions) could be promising sources of active components against the epimastigotes of T. cruzi.
The anothers would like to thank Dr. Hossein Akhani for collection and identification of all of Achillea plant samples and Dr. Vali-allah Mozaffarian for identification of Satureja species.
Ambrozin ARP, Vieira PC, Fernandes JB, Fernandes da silva MFG and de Albuquerque S. Trypanocidal activity of Meliaceae and Rutaceae plant extracts. Mem.Inst. Oswaldo cruz (2004) 99: 1-5
Coura JR and de Castro SL. A critical review on Chagas disease chemotherapy. Mem. Inst. Oswaldo cruz (2002) 97: 3-24
Nogueda- Torres B, Rodriguez- Paez L, Ramirez IB and Ramirez CW. Trypanocidal activity of isopropyl salicylaldehyde and 4- isopropyl salicylic acid on Trypanosoma cruzi. Rev. Lat. Microbiol. (2001) 43: 1-6
Zargari A. Medicinal Plants. 5th ed. Tehran University Publication, Tehran (1992) 528
Rustaiyan A, Masoudi S and Yari M. The essential oils of Achillea aucheri Boiss. and A. Kellalensis Boiss. et Hausskn. From Iran. J. Essent. Oil Res. (1999)11: 19-20
Huber-Morath A. Achillea. In: Rechinger KH. (ed.) Flora Iranica. No. 158, Ackademiche Druck-U. Verlagsansfalt. Graz (1989) 57-58
Rechinger H. Flora Iranica, Labiatae. No.150, Akademische Druck Verlagsantalt. Graze (1986) 499-501
Kiuchi F, Itano Y, Uchiyama N, Honda G, Tsubouchi A, Nakajima- Shimada J and Aoki T. Monoterpene hydroperoxides with trypanocidal activity from Chenopodium ambrosoides. J. Nat. Prod. (2002) 65: 509-12
Uchiyama N, Kiuchi F, Ito M, Honda G, Takeda Y, Khodzhimatov OK and Ashurmetov OA. New icetexane and 20 norabietane diterpense with trypanocidal activity from Dracocephalum komarovi. J. Nat. Prod. (2003) 66: 128-31
Uchiyama N, Ito M, Kiuchi F, Honda G, Takeda Y, Khodzhimatov OK and Ashurmetov OA. A Trypanocidal diterpene with novel skeleton from Dracocephalum komarovi. Tetrahedron Lett. (2004) 45: 531-33
Phillipson JD and Wright CW. Medicinal plants against protozoal disease. Trans R. Soc. Trop. Med. Hyg. (1991) 85: 155-65
Saeidnia S, Gohari AR, Uchiyama N, Ito M, Honda G and Kiuchi F. Two new monoterpene glycosides and other trypanocidal terpenoids from Dracocephalum kotschyi. Chem. Pharm. Bull. (2004) 52: 1249-50
Gohari AR, Saeidnia S, Matsuo K, Uchiyama N, Yagura T, Ito M, Kiuchi F and Honda G. Flavonoid constituents of Dracocephalum kotschyi growing in Iran and their trypanocidal activity. Nat. Med. (2003) 57: 250-52
Wollenweber E, Valant- Vetschera KM, Ivancheva S and Kusmanov B. Flavonoids aglycones from the leaf surfaces of some Achillea species. Phytochem. (1987) 26: 181- 82