In vitro antileishmanial effects of antibacterial diterpenes from two Ethiopian Premna species: P. schimperi and P. oligotricha
© Habtemariam; licensee BioMed Central Ltd. 2003
Received: 3 April 2003
Accepted: 6 June 2003
Published: 6 June 2003
Three antibacterial diterpenes: (5R,8R,9S,10R)-12-oxo-ent-3,13(16)-clerodien-15-oic acid (1), 16-hydroxy-clerod-3,13(14)-diene-15,16-olide (2) and ent-12-oxolabda-8,13(16)-dien-15-oic acid (3) were previously isolated form Premna schimperi and P. oligotricha. Since andrographolide and other structurally related diterpenes were shown to have antileishmanial activity, the aim of the present study was to assess the in vitro effect of premna diterpenes against Leishmania aethiopica; the causative agent of cutaneous leishmaniasis in Ethiopia.
The diterpenes showed potent concentration-dependant suppressive effect on the viability of axenically cultured amastigotes of L. aethiopica. The clerodane diterpenes 1 and 2 were most active (LD50 values 1.08 and 4.12 μg/ml respectively) followed by andrographolide and 3. Compounds 1 and 2 appear to be over 20 and 10-times respectively more selective to leishmania amastigotes than the permissive host cell line, THP-1 cells or the promastigotes stage of the parasites.
The clerodane diterpenes (1, 2) which were more potent and selective than labdanes (andrographolide and 3) are promising for further studies and/or development.
Leishmaniasis is a vector-borne disease that is transmitted by sandflies and caused by obligate intracellular protozoa of the genus Leishmania. Current estimates indicate that leishmaniasis affects people in 88 countries, with 350 million at risk of contracting the disease and approximately 2 million new cases reported each year [1, 2]. During the last decade, the geographic region of leishmania as well as incidence of infection significantly increased mainly due to increased urban development and immunosuppressive illnesses such as AIDs [1, 3].
Effects of test compounds on promastigotes viability
Antileishmanial activity of diterpenes (1–4) and AMB. LD50 values in μg/ml obtained from a minimum of three separate experiments are shown.
0.189 ± 0.01 (n = 3)
0.183 ± 0.04 (n = 6)
4.12 ± 0.28 (n = 5)
11.67 ± 0.88 (n = 3)
1.08 ± 0.25 (n = 5)
12.13 ± 1.42 (n = 4)
19.2 ± 2.24 (n = 5)
26 ± 3.89 (n = 4)
18. 33 ± 1.05 (n = 3)
4.52 ± 0.97 (n = 5)
7 ± 1.08 (n = 4)
Effects of test compounds on axenically cultured amastigotes viability
Cytotoxicity of test compounds in THP-1 monocytes
In traditional medicine, the leaves extracts of Premna schimperi are used to treat external injuries and secondary infection in wounds . Previous systematic biological and phytochemical studies on this plant have identified the clerodane diterpene 1 as the antibacterial principle . Subsequent studies on a related species, P. oligotricha, resulted in the isolation of two antibacterial diterpenes, a clerodane (2) and labdane (3) [13, 14] together with various other compounds [15–17]. Primarily due to owing the presence of an α,β-unsaturated moiety, compound 1–3 did also show cytotoxicity to various cancer cell lines [11, 12]. A related labdane diterpene, andrographolide (4, see Fig. 1), is also shown to possess antiproliferative effects in cancer cells  but its non-toxic concentrations are known to possess various other biological activities including antiinflammatory, antiviral and antithrombotic  and cytoprotective effects from toxins [20, 21]. Recently, andrographolide (4) has also demonstrated to have antileishmanial effects in the in vivo model of leishmaniasis . Another antileishmaial diterpenes of close structural similarity with compounds 1–3 are labdane 5  and abietane, 6 (Fig. 1) and its derivatives , which have been shown to induce leishmanial cell death at concentrations below that cause toxicity in mammalian host cells. With the established antileishmanial effects of structurally related diterpenes, the aim of the present study was to assess the in vitro effect of compounds 1–3 against promastigotes and axenically-cultured amastigotes of leishmania parasites.
For various reasons including simplicity in in vitro culture maintenance, routine screenings of antileishmanial chemotherapeutic agents are often based on promastigotes susceptibility assays. The cellular, molecular and biochemical characteristics of amastigotes, responsible for the clinical manifestation of leishmaniasis in humans, however differ from promastigotes . Hence, the chemotherapeutic potential of drugs for leishmaniasis must further be validated using the intracellular-amastigote forms of the parasites. Since anti-amastigote assays using ex vivo cultured macrophages or permissive cell lines has various limitations , recent researches in many laboratories have focussed on the development of in vitro culture conditions for maintenance and bioassays using axenically-cultured amastigotes. For some leishmania species, including the L. aethiopica strain (MHOM/ET/72/L100) used in the present study, temperature elevation alone could result in differentiation of promastigotes to amastigotes [26–28]. Lowering the pH to 5.5 together with temperature elevation and 5% CO2-air mixture have now been widely used not only to differentiate promastigotes to amastigotes but also for their long-term culture [28, 29]. Hence, this well established condition and medium 199  were used in the present study to obtain and maintain L. aethiopica axenic amastigotes. The resulting axenic amastigotes of L. aethiopica could differentiate back to promastigotes by centrifuging and re-suspending them in promastigotes medium (RPMI 1640) followed by incubation at 22°C. Phase contrast microscopic observations revealed the expected loss of flagella, cell rounding, aggregate formation and the metabolic differences as evidenced by the ability of axenic amastigotes to readily reduce alamar blue. While axenically cultured amastigotes were able to reduce alamar blue in 4 h, promastigotes were only weakly able to do so after long term incubation.
In agreement with previous report on the antileishmanial effect of abietane diterpenes , the effect of diterpenes 1–4 was more pronounced in the amastigotes, mammalian-stage of the parasite than the promastigote, insect-stage. On the other hand, the antileishmanial effect of AMB which is known to be mediated through formation of membrane channels permeable to water and ions  and references there in) was not developmental stage specific as both promastigotes and amastigotes were equally susceptible to AMB. The order of potency of diterpenes 1–3 was similar with their antibacterial effects in previous studies [13, 14]:i.e. 2 >1 >3.
It is interesting to note that differences between the toxicity of the compounds studied to axenic amastigotes and the permissive host cells, THP-1 cells were observed. In previous studies, 50–500 μg/ml concentrations of andrographolide (4) were reported as non-toxic to peritoneal macrophages  but the study had limitations as cells were exposed to the drug for only one hour. In the present assay system, the same incubation time period (72 h) as the antileishmanial assay was used so that direct comparison between toxicities to host cell and parasites could be made. Andographolide (4) and the other labdane diterpene (3) appear to be less selective as amastigotes/THP-1 toxicity ratio (see Table 1) was less than two. On the other hand, the amastigotes/THP-1 toxicity ratio for the clerodane diterpenes 1 and 2 were over 20 and 10 respectively. AMB did also show small but significant toxicity at its higher concentrations. The most toxic side effects that limit the clinical use of AMB, however, are neurotoxicity and nephrotoxicity .
In summary, the present study demonstrate potent antileishmanial activity of premna diterpenes (1–3). The compounds were more potent in inhibiting the axenically-cultured amastigotes than promastigotes of L. aethiopica. The clerodane diterpenes (1, 2) with more selectivity to amastigotes than THP-1 cells are promising and warrant further studies and/or development.
Chemicals and reagents
The isolation and characterisation of diterpenes (1–3) have previously been described [13–15]. Andographolide (4) and all tissue culture media and supplements were obtained from Sigma-Aldrich Chemical Company (Dorset, UK). The stock solutions of test compounds were prepared in dimethyl sulfoxide (DMSO) which was subsequently diluted in the appropriate medium. All DMSO concentrations used (less than 1%) did not show any activity in cell viability studies (data not shown).
L. aethiopica (WHO designation: MHOM/ET/72/L100) promastigotes as well as the human monocytic THP-1 cells were cultured with RPMI 1640 medium supplemented with 10% heat inactivated foetal bovine serum (FBS), 2 mM L-glutamine, 50 IU/ml penicillin and 50 μg/ml streptomycin. Cultures were maintained at their optimum temperature, i.e. 22 and 37°C (5% CO2) respectively.
Promastigotes and drug susceptibility assay
For drug susceptibility assays, 106 cells/ml promastigotes were seeded in 96-well flat-bottom plates in final volume of 200 μl. Various concentrations of experimental drugs made in three-fold dilutions were then added to triplicate wells and plates incubated for 72 h at 22°C. At the end of incubation, cell viability was measured by counting the number of motile cells using a haemocytometer. For each drug, the concentration-response curve was plotted from which LD50 values (the concentration of the agent that reduce cell viability by 50% compared to controls) were determined. All experiments were repeated at least three times.
Axenically-cultured amastigotes and drug susceptibility assay
Late stationary phase (3 × 107 cells/ml) promastigotes were centrifuged and re-suspended in medium 199 with Hank's salts supplemented with 20% FBS, 2 mM L-glutamine, 50 IU/ml penicillin, 50 μg/ml streptomycin and the pH titrated to 5.5 using 1 N HCl. Following incubation of cells at 31°C, 5% CO2, amastigote-like rounded morphology together with loss of flagella and cell clumping started appearing within 24 h but these changes were not synchronous for a week as some motile parasites with intermediate forms and short flagella were detected. Drug treatment of the amastigotes was essentially similar with the promastigotes culture described above. Briefly, amastigotes (4 week old following initiation of differentiation) were incubated for 72 h at 31°C, 5% CO2 in the presence or absence of drugs. Alamar Blue™ (20 μl; Serotec, UK) was added during the last 4 h of incubation and viability measured fluorometrically by using Labsystem's Fluoroskan Ascent flourimeter at excitation wavelength of 544 nm and emission at 590 nm.
Assessment of drugs toxicity in THP-1 monocytes
THP-1 monocytes were plated onto 96 well plates at a density of 4 × 104 cells per well (in 200 μl volume) in the presence or absence of experimental agents and plates incubated at 37°C, 5% CO2 for 72 h. After adding Alamar Blue™ during the last 3 h of incubation, cell viability was measured fluorometrically as described above.
The author wishes to thank Dr David Humber of the School of Bioscience (University of East London) for his gift of THP-1 cells and L. aethiopica promastigotes and Mr Geoff Cooper (University of Greenwich) for his assistance in obtaining the photographs of leishmania parasites.
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