Antiprotozoal activity of diospyrin towards Leishmania donovani promastigotes in vitro

738 TRANSACTIONSOF THE ROYALSOCIETYOF TROPI~+LM~DIUNEAND HYGIENE (1987) 81, 738-741

Antiprotozoal

activity of diospyrin donovani promastigotes

towards Leishmania in vitro

AHINDRA K. SAHA’, RATNAMALA RAY’, DURLAV K. ROY**, PRATIMA SUR’ AMALENDU BANERJEE~ Pharmacy, ‘Dept. of Food Technology and Biochemical Engineering and 3Departwmt Chemistry, Jadavput University, Calcutta 700 032, India

BANASRI HAZRA’*, ‘Department

of

AND

of

Abstract

Diospyrin, a bis-naphthoquinone derivative, isolated from a plant, known for its antitumour nronerties anainst Ehrlich ascites carcinoma in Swiss A mice, exhibits antiprotozoal activity towards i. honovan~ promastigotes in culture. Introduction Leishmania donovani, the causative agent of kala-

azar, is still endemic in parts of China and the Indian subcontinent. There is no universally effective nontoxic form of chemotherapy against this protozoan (PEARSON et al., 1982>,which exists in the phlebotomine sandfly as a flagellate extracellular promastigote and in humans as a non-flagellate amastigote in macrophages. Diospyrin, a bis-naphthoquinone derivative (Fig. 1) isolated from the stem-bark of Diosmos momma Roxb., has been shown by HAZRA et al. (1984) to exhibit potential antituxnour activity towards a transplanted tumour strain, Ehrlich ascites carcinoma in vitro and in oivo in mice; its LDso is 25 mg/kg intraperitoneally in Swiss A mice. It seemedinteresting to determine whether diospyrin would also be active against L. donovani, since a number of antitumour drugs are known to exhibit cytotoxicity towards various microorganisms (KRISHNASWAMY & PURUSHOTHAMAN, 1980).

OH

Fig. 1. Structure

0

of diospyrin.

Materials and Methods Isolarion of dimpyrin. Diospyrin melting point 256°C was isolated from the stem bark of DiospyrosmontanaRoxb. and purified as described previously (HAZRA et al., 1984); its *Address for correspondence. + Deceased.

purity was confirmed by comparison of its spectral data and mixed melting point with those of an authentic sample (LOPES et al., 1973). A solution of diospyrin was prepared by maceration of a weighed amount of the compound with Tween-80, followed by solution in a known volume of dimethyl sulphoxide (DMSO; E. Merck, India), and used for the investigations. Culture and maintenanceof L. donovani. The strain used was UR6, originally obtained from the Indian Institute of Chemical Biology, Calcutta. It was grown and maintained at 22 & 1°C on blood-agar slants prepared from a slightly modified Ray’s medium (RAY! 1932), 100 ml of which contained the following ingredients: glucose, A.R., 1.7 g; sodium chloride, A.R., 0.6 g; peptone (Oxoid, England), 1.0 g; Agar (Difco Laboratories, USA), 1.7 g; beef-heart extract, 50 ml; rabbit blood haemolysate, 5 ml. The pH of the medium was 7.2. The logarithmic phase of growth of L. donovani was reached within 72 h of subculture. Preparation of cell suspension.L. donovani cells, collected after 72 h of incubation at 22 f 1°C were suspendedin cold normal saline (O-15M, pH 7.2); the suspension was centrifuged at 5X, 2OOOxgfor 10 min. The cells were washed similarly twice with cold sterile normal saline. Finally, the packed cell pellet was suspended in phosphate-buffered saline (PBS; 0.01 M sodium phosphate buffer, 0.15 M NaCl; pH 7.2) and homogenized on a cyclomixer. The cells were checked under the microscope for motility as an indication of viability before each experiment. The number of cells per ml was determined by counting in a Neubauer counting chamber. The cell suspension was standardized by estimating its total protein content, which was correlated with its optical density and the cell count. The suspension was diluted with PBS to get a protein content of the order of 10 mgiml, which contained approximately 2.7 X 10s cells/ml, showing an optical density of 0.7 at 680 nm. Inhibition of growth of L. donovani cells in presenceof diospyin in liquid culture medium.L. dotwvani promastigotes were grown in a liquid culture medium (CHAUDHURIet al., 1982), 100 ml of which contained the following ingredients: glucose, A.R., 1.0 g; sodium chloride, A.R:, 0.6 g; peptone (Oxoid), 1.0 g; choline chloride (Sarabhat M. Chemicals, India), 0.3 g; folic acid (Glaxo), 0.1 mg; KHzP04, 20 mg; MgS0,7HZ0, 1.0 mg; NaHCOs, 1 mg; haemin (Hoffmann La Roche, Switzerland), 0.1 mg. The pH was adjusted to 7.2. An inoculum of 2 x IO6cells/ml of L. donovaniat their log phase of growth was added to the medium, from which aliquots of 10 ml were taken into 4 setsof sterile tubes. 3 sets of tubes were treated with 3 different concentrations of diospyrin (Fig. 2) after filtration through a Millipore lilter (0.45 v porosity)! while the fourth set was kept as the vehicle control, which was inoculated with the samevolume of DMSO as that used to dissolve the drug added to the treated tubes. The whole operation was carried out aseptically in an ultra-violet cabinet. The tubes were then incubated

739

B. HA2 :xu et al.

at 25°C. Aliquots were drawn from each of the tubes at definite intervals and the average growth was measured by counting the cells as described above. Measurementofrespiration of L. donovani cells.The oxygen uptake of the L. donovani cell suspension (10 mg/mf of cell protein) in PBS was measured in a Warburg respirometer in the presence of known concentrations of diospyrin solutions (HAZRAet al., 1984), following the conventional manometric techniques (Fig. 3). Respiration was stimulated by adding glucose (2 mi+t)to the buffer. Control experiments were run concurrently in the presence of the same volume of DMSO (the drug vehicle). Measurementof the releaseof intracellular material from L. donovani in thepresenceof diospyrin (CHATTOPADHYAY et al., 1977). From a cell suspension of L. donovani in PBS, 1 ml aliquots were taken into 2 seriesof tubes. The first serieswas kept as a control. A known amount of diospyrin solution was added to each of the tubes in the second series and the final volume in each tube was made up to 3 ml with PBS. The tubes were incubated at 37°C. One tube from each serieswas taken at intervals of 30 min and centrifuged at YC, 5OOOxg, for 10 min. The supematant (cell free under the mrcroscope) was removed and suitably diluted with PBS. The absorbance was measured at 260 run and 280 nm with a Beckman (DU) spectrophotometer against an appropriate blank. A parallel experiment was conducted by addition of cold perchloric acid (0.5 M) instead of the drug, in order to get an estimate of the total pool of proteins released at the respective wavelengths. Each of the above experiments were repeated 3 times.

Results

Growth inhibition in liquid culture medium (MARR t3 BERENS, 1977). After 72 h incubation in liquid culture medium luxuriant growth was observedin the

control tubes (1.8 x 10’ cells/ml), while the tubes containing 1 uglml of diospyrin did not contain any living cell, indicating total inhibition of growth. In a separate experiment involving periodic monitoring using different concentrations of diospyrin, a striking growth-inhibitory effect of the compound was observed, as shown in Fig. 2, representing the average of 3 sets of experiment. The minimum inhibitory concentration was 1 ug/ml for diospyrin and 2 uglml for pentamidine, an antileishmanial drug in current use. Inhibition

of respiration in presence of diospyrin.

Studies on the kinetics of oxygen uptake by L. donovani cells in the presenceof diospyrin revealed a considerable respiratory inhibition. After 100 min incubation of L. donovani cells in 1.0 and 2.5 ug/ml of diospyrin, the endogenousrespiration with respect to the control was inhibited by about 42% and 80% respectively, while the glucose-stimulated respiration was inhibited by 38 and 67% respectively, with the same drug concentrations. (Fig. 3). At a concentration of 5 ugiml, all Or consumption by the organism was stopped within approximately 60 min of contact

8.00

I 10

I 20

I 30

I 50

I LO Time

(Hours

I 60

I 70

I 80

I 90

1

1

Fig. 2. Inhibition of growth of L. dmwoani cells in liquid culture media contaiohg diospyrio at various concentrations (meansof 3 experiments). n control (without diospyrin); 0 0.5 <d of diospyrin; 0 0.8 &ml of diospyrin; A 1.0 &ml of diospyrin; A 2.0 pg/ml of pentamidine.

740

%

ANTIPROTOZOAL

ACTIVITY

OF DIOSPYRIN

TOWARDS

L. dotwvani promastigotes in vitro

90

E” &) 75 a z 5.560 @ 0, 0 “Q c-45 g 5 z 6 5 0’ c m

30

15

0

20

X

40

60 Time ( Mins >

80

100

Fig. 3. Inhibition of glucose-stimulated respiration of L. dowwni cells in the presence of diospyrin at various concentrations, measured at 37°C. 0 Endogenousrespiration of L. dowvani cells in PBS medium; A Endogenous + drug vehicle (DMSO); n Endogenous + diospyrin (1.0 I.Lg/ml); 0 Endogenous + diospyrin (2.5 &ml); 0 Endogenous + gl
Table-Release

of 260 nm and 280 nm absorbing

Tube No.*

Incubation system

1

Cell + Cell + Cell + CeU + Cell + Cell +

: 4 5 6

PBS PBS + vehicle PBS + drug (I .O &ml) PBS + drug (2.5 &nl) PBS + drug (5 &ml) 0.3 M perchloric acid

intracellular

0 0.068 0.070 0,070 0.071 0.072

materials

from L&mania

donarani in presence of diospyrin

Absorbance per mg of cell protein at times shown (min) 260 nm 280 nm 30 60 90 120 0 30 60 90 0.068 0.070 0.080 0.120 0.170

0.069 0.075 0.110 0.220 0.310

0.069 0.076 0.130 0.250 0.350

0.070 0.078 0,150 0.270 0.380 0.700

0.058 0.060 0.060 0.060 0.065

0,058 0.060 0.060 0.070 0.100

0.058 0.060 0.090 0.120 0,160

0.059 0.062 0.100 0.140 0.200

120 0.059 0.063 0.110 0.160 0.220 0.4Oll

*Tubes No. 1 & 2 served as controls. Tube No. 6 measuredthe absorbancefor the total amount of intracellular material present in the cells in each tube. Each set of data represents the average of 3 sets of experiments.

with the drug (data not shown). Under similar experimental conditions, 40 uglrnl of pentamidine effected about 80% inhibition. Release of intracellular material from L. donovani cells in the presence of diospyrin. Appreciable leakageof intracellular material took p$e from the cells of L. donovani incubated with didspyrin at concentrations of 1.0, 2.5 and 5-O ug/ml (Table). The process of release continued over a period of 120 min. The releaseof such intracellular material from the protozoan cells during this period in the absence of the drug was negligible (Table 1). Microscopic observations. An L. donovani cell-suspension, treated with 5 ug/ml of diospyrin, was observed microscopically within 2-3 h of drug contact. The untreated cells were similarly observed and

found to be mostly motile, long and slender, with their moving flagella projecting (Fig. 4). The treated cells were visibly distorted, showing no distinct boundary; all were swollen, non-motile and without any flagellum, while some were fragmented (Fig. 5). After 3 h most of the cells were lysed. Discussion These results vividly demonstrate the antiprotozoal activity of diospyrin. Currently, the mainstays of chemotherapy available for the 3 major forms of leishmaniasis are pentavalent antimonial compounds (BERMAN & WYLER, 1980), followed by pentamidine and amphotericin B, which often require careful parenteral administration (NEVA et al., 1979). The need for development of a non-toxic and easily

8.

HAZRA

Fig.4. Photomicrograph (X1000)of normalcellsof L. donovnnr after 2 h incubatmnat roomtemperamre. administered drug cannot be over-emphasized. Studies of the pharmacology of anti-leishmanial agents have been seriously hampered by the general insensitivity in vitro of the cultured promastigotes to various leishmanicidal drugs (BEVERAGE, 1963). Several experimental models have been proposed to facilitate such studies (BERMAN& WYLER, 1980.;MATTOCK & PETERS,1975); the in vitro susceptibibty of cultured L. donovani towards diospyrin is therefore quite significant. The growth of cells in liquid culture media was totally stopped in the presence of diospyrin at such a

low concentration as 1 ngiml, while almost total inhibition of respiration was observed with 5 rig/ml of

diospyrin. Continuous leakage of metabolites absorbing at 260 nm and 280 nm from the cells incubated with diospyrin indicated a change in the permeability of the cells, caused by the drug. The apparent anomaly between the minimum inhibitory concentration (MIC) on the one hand and the drug concentration for respiratory inhibition and for metabolite release on the other hand may be attributed to the difference in period of contact in these 2 sets of experiments. MIC is a reflection of the inhibition of cell growth and cell division; the drug can exert its effect over a prolonged period of time by a direct or indirect interference with the biosynthetic machinery of the cell. On the other hand, effects on respiration and release are short-term, when the direct effect of the drug is observed in terms of depletion of metabolites of the internal pool. More elaborate studies are needed to elucidate the mechanism of action. The substantial loss of morphological characteristics of the cells incubated with diosuvrin confirms its leisbmanicidal activity. Acknowledgements We are grateful to the University Grants Commission and the Indian Council of Medical Research, for financial support to B.H. and R.R. respectively. We thank Prof. A. Correia Alves, Faculty of Pharmacy, University of Oporto, Portugal, for a sample of authentic diospyrin. The generous help of Mrs G. Raymahasay is gratefully acknowledged. Someof this work has been submitted by B. Hazra in partial

et

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al.

Fig. 5. Photomicrograph (XlooO)of treatedcellsofL. dowant after 2 h incubation with 5 p&l of diospyrinat room temperature,

indicatingswelling,distortionandlossof intracellularmaterial. fulfilment of the requirements for a PhD degree in the Faculty of Science, Jadavpur University. References Berman, J, D. & Wyler, D. J. (1980). An in vitro model for investigation of chemotherapeutic agents in leishmaniasis. 3oum5I of Infectious Diseases, 142, 83-86. Beverage, E. (1963). Chemotherapy of leishmaniasis. Experimental Chemotherapy, 1, 257-287. Chattopadhyay, T. K., Ghatak, S. N. & Bhaduri, A. N. (1977). Antileishmanial activity of mycobacillin. Journal of Antibiotics, 30, 262-266. Chaudhuri, G., Chatterjee, T. K. & Banerjee, A. B. (1982). Growth factor requirements for in vitro growth of L. donovani. Indian Journal of Medical Research, 76, 157-

163. Hazra, B., Sur, P., Roy, D. K., Sur, B. & Banerjee, A. (1984). Biological activity of diospyrin against Ehrlich ascites carcinoma in Swiss A mice. Planta Medica, 51, 295-297. Krishnaswamy, M. & Purushothaman, K. K. 11980). Plumbagin; a study of its anticancer, antibacterial and antifungal properties. Indian Journal of Experimental Biology,

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Marr, J. J. & Berens, R. L. (1977). Antileishmanial effect of allopurinol IL Relationship of adenine metabolism in Leishmania speciesto the action of allopurinol. Journal of Infectious Diseases, 136, 724-732.

Mattock, N. M. & Peters, W. (1975). The experimental chemotherapy of leishmaniasis II. The activity in tissue culture of some antiparasitic and antimicrobial compounds in clinical use. Annals of Tropical Medicine and Parasitology, 69, 359-371. Neva, F. A., Wyler, D. & Nash, T. (1979). Cutaneous leishmaniasis - a case with persistent organisms after treatment in presence of normal immune response. American Journal 467-47 1.

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Pearson, R. D., Martian, A. A., Harcus, J. L., Hall, D. & Hewlett, E. L. (1982). Lethal effect of phenothiazine neuroleptics on pathogenic protozoan Leishmania donovani. Science, 217, 369-371. Ray, J. C. (1932). Cultivation of Leishmanro parasites on solid medium. Indian Journal of Medical Research, 20, 355-367. Accepted for publication

16 December 1986