Evaluation of Plant Extracts for Antileishmanial Activity using a Mechanism-Based Radiorespirometric Microtechnique (RAM)

in vitro Gongronema latifolia, Dorstenia multiradiata, Picralima nitida. Cola attiensis Desmodium gangeticnm Leishmania


Introduction
Infections due to protozoa of the genus Leish,nania are a major world wide health problem, with high endemicity in developing countries. The global prevalence of leishmaniases in man is about 12 million cases, with an estimated incidence of 2-3 million cases per annum (1, 2). Approximately 350 million people within 80 countries are threatened by the disease worldwide. The pathological effects of the disease are complex, manifesting in various forms. They include self-healing cutaneous lesions, recurrent leishmaniasis recidivans, disfiguring mucocutaneous and diffuse cutaneous diseases, and visceral leishmaniasis (kala azar). In the advanced form of the latter, the reticuloendothelial system is infected with the resultant toll on the spleen, liver, bone marrow, lymph glands, and, often, some degree of intestinal tract disfunction. The mucocutaneous, diffuse cutaneous, and visceral disease forms can be fatal if untreated. Development of a new drug for the treatment ofleishmaniasis has been impeded by the lack of a simple, rapid and universally applicable (i.e., to the various Leishmania species/strains infecting humans) drug evaluation system (8,9). The lack of progress in the development of new antileishmanial agents is evident by the fact that all the clinically useful drugs were developed between 1947 and 1959(9). Current methods used for the screening of potential antileishmanial agents generally utilize intracellular amastigotes (the mammalian intracellular form) since promastigotes (monoflagellate forms found within the insect vector and culture in vitro) are reported "insensitive" within in vitro assays to SbV compounds used for human leishmaniases (9). Since there is no system yet available for culturing amastigotes extracellularly, except re-isolation from infected tissues and macrophage cultures, their mass culture is rather limited (8,9), making them unsuitable for primary screening of potential antileishmanial agents.
An in vitro radiorespirometric microtest (RAM) using promastigotes has been developed in our laboratories which relies on drug inhibition of parasite production of 14C02 from a battery of '4C-substrates by promastigotes to detect drug-mediated parasite damage at low drug concentration within a short time (10. ii). The test is quantitative, rapid, consistent, and conducted in a serum-free chemically-defined medium in which prior adaptation is not necessary to cultivate the so-called "difficult to grow" species. The method has been shown to correlate well with patient response to SbV therapy (11).
Visceral leishmaniasis is endemic to the central Nigerian highlands, and zoonotic cutaneous leishmaniasis, prevalent in the northern half of this country (12). Because of limited supply, expense and high toxicity of commercial antileishmanials, traditional herbal therapy is frequently utilized in many leishmanial endemic regions of Nigeria.
In this study, we have evaluated extracts of 11 plants used in Nigerian folk medicine as antiparasitic remedies for possible antileishmanial activity using the radiorespirometric microtest. RAM.

Extraction procedure
Two hundred grams of powdered material from each plant were percolated for 24 hours with 80% methanol or dichloromethane and concentrated to a sticky gum under reduced pressure. The extracts from the seed materials were partitioned between chloroform and water and the two fractions were submitted to bioassay. The list of extracts prepared is shown in Table   1.
Cultivation ,nediu,n Proinastigotes of L. chagasi were grown in a serum-free, defined medium. MM2 (10). The MM2 medium contained l2Opg/ml protein ll0;g/mI human transferrin. l0ig/ml human insulin. 100 ig/ml defatted bovine albumin), plus lOpg/ml low density bovine lipoprotein. Previous research demonstrated the need for low protein-serum-free medium because serum protein drug association reduces in vitro antiparasite activity (10). Cultures were maintained at 25C during growth and incubation with drug.

Radiorespirometric procedure
Promastigotes were maintained in log phase growth for 3 successive transfers (48-72 h apart) prior to radiorespirometric testing. Test extracts (or PBSS plus drug solvent [DMSOI, for parallel control cultures) were added 24h after the third promastigote transfer to fresh growth medium. Incubation in the presence of plant extracts was continued for 96 additional hours while the parasites remained in mid-log phase growth. The rest of the radiorespirometric procedure was conducted as previously described (10). Briefly, to each well of a microtiter tray (Biospherics Type TOlO + COlO, Universal Plastics & Engineering Company, Rockville. Maryland) were added 25M' of a single '4Csubstrate (100,000 dpm). The tray was covered with a friction-fit lid to prevent evaporation while the promastigotes were being 3 x centrifugally (700 x G. 10mm, 4°C) washed free of nutrient medium and drug using PBSS. The final organism pellet was resuspended to a concentration of I x io organisms/mi in PBSS.
After the addition of 25Ml of organism suspension to each well (total volume per well, 5OfLl: 14C-substrate + promastigote suspension), the wells were immediately covered with a filter paper disc (22 mm, * 410, Schleicher & Schuell, Inc., Keene, New hampshire) which had been premoistened with one drop of saturated Ba)Ohl) solution. The trays were recovered with the lid. 11 during the 3() mm incubation at 33°C the I.eish,nania metabolize the '4C-substrates to '4C05. the radioactive gas was collected as a Bai4CO,i precipitate on the filter paper discs. After the incubation. the filter discs were removed from the trays, dried using an infrared lamp. and the '4C quantity determined using an argon methane (PlO mixture, argon: methane 9: 1 v/v. respectively) gas-flow proportional counter (Model 5110. Tennelec. Inc.. Oak Ridge. Tennessee). Data (dpm corrected for background. I count per minute: and machine efficiency) were electronically sent to a computer for analysis and graphic presentation. \ quantitative replicate test variability was determined in a previous study (10). Tests were initially repeated in duplicate on 4-5 separate days (8-10 tests/drug concentration/organism). The mean dpm!14C-substrate had a linear relationship to the magnitude of the standard deviation 1St)) (1(t). It was established from the analysis of previous data on the test system that the percent coefficient variation is estimated to be approxiniatelv 19 and 15% at counting levels of 100 dpm and 20.00(1 dpm. respectielv lit)). Therefore testing was only repeated in duplicate for each test extract with parallel duplicate drug vehicle control tests (unless otherwise indicated).

Drug lest procedure
The procedure was conducted as described earlier (10). The extract concentration of 5Oig/rnl was used for the tests. Drug activity was based on determining the '4C substrate(s) ('fable 2) for which '4C0, release was decreased in the drug-treated parasites compared to the effect on the control group.  make drug solution). Since there were no parasites in the nonbiological control, any 4COu detected s as attributed either to biologic contamination or, less likely, chemical conta niination el the '4C-substrates resulting in breakdown of the '4C-substrates. II' radioactivity above background 110 clisintegrations per minute, dpm) was detected in the nonbiological control. the suspect solution)s) was replaced and the experiment was repeated. At a concentration of 50 pg/mI. 5 of the plant extracts tested inhibited the catabolism of two or more of the substrates to CO (Table 2). C. attiensis extract (CT) inhibited parasite catabolism of 10 of the 21 substrates used in the assay. with the strongest activity observed on the disintegration of L-ornithine. -proline. asparagine, and -aspartic acid (Fig. 1). G. latifolia (GG) displayed strong inhibition of the catabolism of succinic and 1)-glucosamine. as well as -proline, L-ornithine, and glutamic acid (Fig. 2). For P. nitida extract (HB). the strongest activity was observed against Na butyric acid. with the drug treated parasite cultures showing a suppression of more than 90% when compared with the values observed for the controls. An inhibition rate of 40 % or more was observed for succinic acid. glycine, and glutamine. Strong activity was also noted for i.-aspartic acid, -glutamic acid, and ornithine (Fig. 3). No significant inhibition occurred in the catabolism of tyrarnine. taurine. The extract of D. multiradiata (1)L) strongly inhibited the catabolism of -ornithine, butyric acid, and proline (Fig. 4). Moderate inhibition was observed on aspartic acid, 1-asparagine. n-mannose. and 1)-galactose. I). gangeticum extract (SM) showed strong inhibition of 5 of the 17 substrates used in the study, with the strongest in-  Test results for the plant extract, SM-7, from Desmodium gangeticum. At 50 ig/ml suppression of parasite catabolism of 9 of 17 '4C-substrates occurred.

Discussion
One of the plants investigated., C. attiensis, is used among other things for the treatment of migraine. bronchitis, and catarrh. P. nitida has been employed in the treatment of malaria, African sleeping sickness, and bacterial infections. D. gangeticum is reputed in folk medicine as a very effective antifungal agent, antiviral, anti-inflammatory, and as an oral remedy for various parasitic skin infections. Aqueous decoction of D. multiradiata is used as an antiviral agent and as a local antiinflammatory. G. latifolia is valued as a bitter tonic, and the alcoholic infusion is dispensed for bilharzia, viral hepatitis, and as a general antimicrobial agent.

Pentavalent antimonials have a serum
half-life of 2 hours with the maximum achievable serum level of approximately 2Ozg/ml Sb (or approximately 73g/ml drug) (13, 3). It is interesting to note that even as crude mixtures, the 5 active plant extracts (Table 3, Fig. 1-5) were active at 5Og/mt and one, DL-55, retained antileishmanial activity to 5 tg/ml. The observation that the crude extracts exhibited antileishmanial activity, at drug concentrations comparable to Shy, seems to indicate high potential for the active drug principles as new antileishmanials.
The plants are presently being analyzed for their chemical constituents. A literature search, however. revealed that the plants vary widely in their constituents.