Alkaloids of picralima nitida used for treatment of protozoal diseases

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention pertains to extracts of Picralima nitida seeds, fruit rind and stem bark, and the use of these extracts in the treatment of malaria, leishmaniases and trypanosomosis.

The alkaloid extracts of the fruits of P. nitida exhibit activity against drug-resistant and drug-sensitive malarial strains of Plasmodium falciparum and these alkaloids show significant inhibitory activity against both clones of P. falciparum at IC.sub.50 values of 0.0.1-0.9 .mu.g/ml.

The invention also pertains to the use of methanol extracts from Picralima nitida for use in the treatment of leishmaniases.

2. Description of the Prior Art

Picralima nitida (Fam. Apocynaceae) is the source of the bitter tasting "Akuamma" seeds, employed extensively in west Africa as an ingredient in many folk remedies.sup.1,.sup.2. The aqueous extract of the bark and seeds are used for the treatment of malaria and pyrexia, and the powdered seeds have been dispensed as a cure for pnumonia and other infections.sup.3.

The plant Picralima nitida contains several indole and dihydroindole alkaloids, of which the major ones include akuammiline, akuammidine, akuammine, akuammigine, akuammicine, picraline and picraphylline.sup.4,.sup.5. The principal alkaloid found in the plant, akuammine, has been shown to be inactive against avian malaria and in clinical trials.sup.6.

Akuammine, however, is a strong sympathicomimetic and possesses local anesthetic action comparable to that of cocaine.sup.7.

Another major Picralima alkaloid, akuammidine has been shown to possess a strong local anaesthetic action and was found to be three times as active as cocaine hydrochloride.sup.8. The compound also has sympatholytic and a mild, but persistent, hypotensive effect. Extracts of the plant have been shown to posses significant analgesic activity in the rat pedal model.sup.9. The hot water decoction of the stem bark has been shown to possess significant in vivo activity against Trypanosoma brucei in rats, and the activity was found comparable to the effect of 8 mg kg.sup.-1 of dimenazene aceturate (Wosu and Ibe, 1989). A CNS active indole alkaloid, pericine, has been detected by opiate receptor binding studies from the cell suspension culture of P. nitida.sup.10. Although seeds and stem bark of P. nitida are employed as aqueous ethanol (palm wine) decoctions in the treatment of severe cases of malaria in Nigeria, Ghana and many parts of Africa, there is presently no scientific investigation to support the use of the herb as a malaria remedy.

Infections due to protozoa of the genus Leishmania are a major world-wide health problem, with high endemicity in developing countries, however, the global prevalence of leishmaniases in man is about 12 million cases, with an estimated incidence of 2-3 million cases per annum.sup.11. The pathological effects of the disease are complex and manifests in various forms ranging from self-healing cutaneous lesions; recurrent leishmaniasis recidivans; disfiguring mucocutaneous and diffuse cutaneous diseases; to fatal systemic infection, visceral leishmaniases or kala azar. In the later form, the reticuloendoethelial system is infected with the resultant toll on the spleen, liver, bone marrow, lymph glands, and, often, some degree of intestinal tract dysfunction. Approximately 350 million people within 80 countries are threatened by the disease worldwide.

Unfortunately, clinical drug intervention is presently limited to the use of pentavalent antimonials (SbV), sodium stilbogluconate and N-methylglucamine antimonate, and, secondarily, amphotericin or pentamidine.sup.12,.sup.13. These antileishmanials require parenteral administration with clinical supervision or hospitalization during treatment because of the severity of possible toxic side-effects that include cardiac and/or renal failure.sup.14.

Treatment with the aforementioned agents is not consistently effective particularly for the most virulent leishmanial disease forms.sup.15,.sup.16,.sup.17,.sup.18. The World Health Organization has reported large scale resistance of kala azar to SbV, which are the preferred chemotherapy for treatment of most forms of leishmanial disease (TDR News, Dec., 1990). In some endemic regions, it has been observed that prolonged medication (22 months or more) with SbV is required to effect a clinical cure.sup.19. Long term SbV therapy, however, is not usually advocated due to the mentioned cardiac and renal toxicity of SbV.

There is, therefore, a need for the development of more effective, less toxic and orally active antileishmanial agents. However, development of a new drug for the treatment of leishmaniasis has been impeded by the lack of a simple, rapid and universally applicable (to the various Leishmania species/strains infecting humans) drug evaluation system.sup.20,.sup.21. 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.sup.22. Current methods for screening 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.sup.23. 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.sup.24,.sup.25, thereby making them unsuitable for primary screening of potential antileishmanial agents.

An in vitro radiorespirometric microtest using promastigotes has been developed which relies on drug inhibition of parasite production of .sup.14 CO.sub.2 03 +3* U .sup.W Etery of .sup.14 C-substrates by promastigotes to detect drug-mediated parasite damage at low drug concentration within a short time.sup.26,.sup.27. 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 to patients response to SbV therapy.sup.28.

Visceral leishmaniasis is endemic to the central Nigerian highlands, and zoonotic cutaneous leishmaniasis, prevalent in the northern half of this country. Therefore, because of limited supply, expense and toxicity of commercial antileishmanials, traditional herbal therapy is frequently utilized in many leishmanial endemic regions of Nigeria.

SUMMARY OF THE INVENTION

It is an object of the invention to procure extracts of Picralima nitida seeds, fruit rind, and stem bark and utilize these extracts for anti-malarial activity or inhibitory activity against drug-resistant clones of Plasmodium falciparum.

A further object of the invention is to provide water, methanol or dichloromethane extracts of Picralima nitida seeds, fruit rind and stem bark for anti-malarial activity or inhibitory activity against drug-resistant clones of Plasmodium falciparum.

A yet further object of the invention is to provide water, methanol or dichloromethane extracts of alstonine, akuammine, akuammicine, melinonine, picraphylline, picraline, and pseudo-akuammigine isolated from the fruits and stem of Picralima nitida as active constituents or ingredients for anti-malarial activity or inhibitory activity against drug-resistant clones of Plasmodium falciparum.

A still further object of the invention is to provide dimers (compounds formed from the combination of isolates of alstonine, akuammine, akuammicine, melinonine, picraphylline, picraline, and pseudo-akuammigine) for example, serpentinine, as active constituents or ingredients for anti-malarial activity or inhibitory activity against drug-resistant clones of Plasmodium falciparum.

A further object still of the invention is to provide methanol and aqueous extracts of Picralima nitida to provide inhibition of leishmania promastigotes.

A yet further object of the invention is to provide methanol and aqueous extracts of Picralima nitida which are sufficiently active at certain concentrations against visceral Leishmania chagasi and cutaneous L. mexicana.

A further object still of the invention is to provide the indole alkaloids akuammine, pseudo-akuammigine, picraline, alstonine and akuammicine isolated from the active fraction for inhibition of leishmania promastigotes.

A still further object of the invention is to provide extracts and isolates from Picralima nitida and Dorstenia multiradiata for treatment of trypanosomiases where other chemotherapeutic agents are generally unsatisfactory due to very high toxicity of these other chemotherapeutic agents or the drug resistance of Trypanosoma brucei.

A further object still of the invention is to provide methanol and aqueous extracts of Picralima nitida seeds or Dorstenia multiradiata to provide antitrypanosomial activity.

A still further object of the invention is to provide the indole alkaloids akuammine, pseudo-akuammigine, picraline, alstonine and akuammicine isolated from the active fraction of P. nitida and anthocyanidins as the active components of the extract from D. multiradiata as agents for antitrypanosomial activity.

Anti-malarial activity using water, methanol or dichloromethane extracts of Picralima nitida seeds, fruit rind and stem bark is obtained against drug-resistant clones of plasmodium falciparum at dosages between about 1.23 to 32 .mu.g/ml.

Inhibition of leishmania promastigotes is accomplished by using methanol and aqueous extracts of Picralima nitida. By using radiorespirometric microtests based on in vitro inhibition of catabolism of 14/CO/2 of a battery of 14/C-substrates by promastigotes, these extracts are active at concentrations of 50 .mu.g/ml or less against visceral Leishmania chagasi and cutaneous L. mexicana. These extracts significantly inhibited (10%-90%) of the catabolism of certain sugars, amino acids, or fatty acid precursors by promastigotes. The indole alkaloids akuammine, pseudo-akuammigine, picraline, alstonine and akuammicine were isolated from the active fraction; however, the greatest inhibition is with alstonine. Alstonine exhibited a dose related activity with the highest growth inhibition being at 50 .mu.g/ml. At 20 .mu.g/ml the alstonine compound showed a growth of 69.3% after 96 hours.

Extracts from Picralima nitida and Dorstenia multiradiata are active at very low doses in the treatment of trypanosomiasis by using a dose of 50 mg/kg and 5 mg/kg of the methanol and aqueous extracts respectively of Picralima seeds. These doses completely cleared animals of the parasites at post-treatment day 12 in rats and day 10 in a mouse model. Methanol extract of Dorstenia gave similar results at treatment day 10 and 8 for the rat and mouse models respectively. The indole alkaloids akuammine, pseudo-akuammigine, picraline, alstonine and akuammicine were isolated from the active fraction of Picralima nitida, whereas anthocyanidins were the active components of the extract from Dorstenia multiradiata.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a thermal spray liquid chromatogram of the dichloromethane extract of P. NITIDA fruit rind.

FIG. 2 shows a thermal spray liquid chromatogram of the dichloromethane extract of P. NITIDA seed.

FIGS. 3(A),(B),(C),(D) show a radiorespirometric microtest based on in vitro inhibition of catabolism of .sup.14 CO.sub.2 of a battery of .sup.14 CO.sub.2 substrates by promastigotes used to examine extracts of plants for antileishmanial activity; wherein Cola attiensis extract (CT) inhibited parasite catabolism of 5 of the 21 substrates used in the assay, with the strongest activity observed on the disintegration of ornithine, L-proline, L-aspartic acid.

FIGS. 4(A),(B),(C) show a radiorespirometric microtest based on in vitro inhibition of catabolism of .sup.14 CO.sub.2 of a battery of .sup.14 CO.sub.2 substrates by promastigotes, used to examine extracts of plants for antileishmanial activity; wherein Gongronema (GG) displayed strong inhibition of the catabolism of succinic acid, D-galactose, D-mannose, L-aspartic acid, L-glutamine and D-glucosamine, as well as L-proline, Na-n-butyric acid, and L-glutamic acid. The GG-8 is highly active against the etiologic agent of visceral leishmaniasis, Leishmania (Leishmania) chagasi.

FIGS. 5(A),(B),(C) depict the activity for succinic acid, glycine and aspartic acid, where an inhibition rate of 40% or more is obtained with glutamic acid, glutamine and methionine.

FIGS. 6(A),(B),(C) show the extract of Dorstenia strongly inhibited the catabolism of ornithinie, butyric acid and mannose.

DETAILED DESCRIPTION OF THE INVENTION

Materials and Methods

P. nitida fruits were collected from trees at a homestead in Anambra state, Nigeria. The seeds and the fruit-rind were separated and air-dried. The stem bark was obtained from the branch of a tree at the above location. Each plant part was cut into small pieces and powdered.

Extraction

Powdered seeds of P. nitida (500 g) were extracted with 5 CH.sub.2 Cl.sub.2 in a Soxhlet extractor for 10 hours. The seeds was air dried and re-extracted with 5 1 of MeOH for about 6 hours. A fresh sample of the seeds (200 g) was extracted with boiling water for 6 hours. The extracts were filtered and concentrated to dryness under reduced pressure, and the aqueous fraction was freeze-dried. The seed oil was obtained from the petroleum ether (b.p. 40.degree.-60.degree.) extraction of the seeds. The fruit rind (200 g) and the stem bark (100 g) were similarly extracted with CH.sub.2 Cl.sub.2 and MeOH.

Alkaloid fractionation

The MeOH extract (10 g) of the stem bark (prepared as described above) was concentrated to a sticky gum and re-extracted (for 30 minutes) with 200 ml of 10% HCl. The aqueous acidic extract was filtered, made alkaline to pH 9 with concentrated NaOH solution and extracted with 10.times.200 ml CH.sub.2 Cl.sub.2. The organic layers were concentrated to dryness under reduced pressure to yield the alkaloid fraction, which were found to contain several Dragendorff positive spots on TLC. The MeOH extracts of both the fruit rind and the seeds were similarly treated and the combined organic layers were concentrated under reduced pressure.

Authentic samples or reference compounds of akuammine, picratidine, akaummigine and akuammiline (from the University of Science and Technology, Kamasi Ghana), picraline, echitamine, akuammicine and .psi.-akuammigine (from Universite de Reims, Champagne-Ardenne, Reims, France), and echitamine (from Laboratoire des Plantes Medicinales du C.N.R.S., B.P. 643 Noumea, New Caledonia) were used as reference compounds.

Antimalarial screening

The in vitro assays were performed by using a modification of the semi-automated microdilution technique described earlier in Desjardins et al..sup.29 and Milhous et al..sup.30 Two P. falciparum malaria parasite clones, designated as Indochina (W-2) and Sierra Leone (D-6), were utilized in susceptibility testing. The W-2 clone is resistant to chloroquine, pyrimethamine, sulfadoxine, and quinine, and the other clone is resistant to mefloquine. The test extracts were dissolved in dimethylsulfoxide (DMSO) and serially diluted with media. The uptake of tritiated hypoxanthine was used as an index of inhibition of parasite growth.

Liquid Chromatography--Mass Spectrometry of P. nitada Extracts

The separation of the constituents of the extracts was conducted on a Varian 5500 liquid chromatograph with a Vista detector. Waters Bodapak C.sub.18 columns were eluted with CH.sub.3 CN--H.sub.2 O (60:40).

Thermospray liquid chromatography-mass spectrometry (LC-MS) was conducted on a Waters liquid chromatograph interfaced to a Nermag R10-10C quadrupole mass spectrometer equipped with a Nermag thermospray source and Vestec thermospray probe and gradient controler. Data acquisition was by Finnigan Super INCOS data system. The thermospray source was operated at 200.degree. C. with the thermospray probe run at T.sub.1 =105.degree. C. and T.sub.2 =190.degree. C. No filament, repeller, or discharge current was applied.

The in vitro antimalarial activities of various extracts of P. nitida on P. falciparum clones are shown in Table 1. All the extracts inhibited the uptake hypoxanthine by the plasmodia at low concentrations, with IC.sub.50 values ranging from 1.23 .mu.g/mL to 32.16 .mu.g/mL. The CH.sub.2 Cl.sub.2 extracts (1,4,6) showed the strongest activity when compared to the methanolic (2,5) and aqueous (3) extracts with IC.sub.50 of 1.61 .mu.g/ml and 5.15 .mu.g/ml for the fruit rind and seeds, respectively, in the W-2 plasmodium clone; and 5.03 .mu.g/ml and 2.4 .mu.g/ml for the respective plant parts in the D-6. The fruit rind (extract 4) had the best activity in the W-2 system while the alkaloidal fraction of MeOH extract (9) of the stem bark gave the best activity in the D-6 system.

The retention times and major ions obtained from the LC-MS of the CH.sub.2 Cl.sub.2 extracts of the seeds and fruit rind, the two most active extracts, are shown in Tables 2 and 3. From the molecular ion peaks obtained, it was observed that the common Picralima alkaloids were not detected as major components of the CH.sub.2 Cl.sub.2 extracts of either the fruit rind or seeds of P. nitida. Akuammine (M+1=m/z 383), .psi.-akuammigine (M+1=m/z 367) and picraline (M+1=m/z 411) were detected in the fruit rind (Table 2). The only peak corresponding to the molecular weight of a known picralima alkaloid, akuammiline was observed at 9:34 min. (M +1=m/z 395) in the LC-MS chromatogram of the seed (Table 3). These constituents occured as minor components of the extracts (FIG. 1 and 2). The LC of SBI and SB2 using authentic samples of Picralima alkaloids indicated the presence of akuammine, akuammicine and traces of akuammigine, akuammidine and picratidine. The retention times of the major constituents of the extracts did not correspond to those of any of the reference compounds. Significant differences were observed in the composition of the rind and the seed extracts (FIG. 1 and 2). LC-MS indicated high mass spectra peaks (>500 m/z) not previously reported from this species, which suggests the possibility of novel compounds being the active components.

Results of the in vitro assay show that the extracts of P. nitida possessed activity against P. falciparum strains. The antimalarial activities of these extracts are superior to those reported for most experimental antimalarial plant and isolates, i.e. Weenen et al.sup.31 ; Khalid et al..sup.32 ; Cubukcu et al..sup.33 The activity of these extracts are apparently weaker (from the relative IC.sub.50 values) than those of the clinically useful antimalarials of plant origin, quinine (cf. Warhurst.sup.34) and artemisinin (cf. Klayman.sup.35). It should be noted, however, that of the test extracts comprised of a mixtureof many compounds, some of the mixtures, in fact, prove to be more active than current antimalarials. It must also be noted that the extracts were active against drug resistant strains of the parasite and this of course indicates a potential for use in cases of drug resistant malaria chemotherapy.

Fractionation of the extracts, using classical alkaloid separation scheme led to significant improvement in the antimalarial activity. The IC.sub.50 value of the stem bark was reduced from 6.46 .mu.g/mL for the crude extract to 2.25 .mu.g/mL in the Draggendorf positive fraction when tested against the W-2 clone, and in the D-6 model, the IC.sub.50 values of 14.86 .mu.g/mL and 1.23 .mu.g/mL were observed for the crude extract and the alkaloid fraction, respectively. While the result suggests the possibility that alkaloids might be active components of this plant, the significant antimalarial activity detected in both seed oil and the aqueous extract indicates a contribution of non-alkaloidal constituents to the anti-malarial activity of Picralima.

It was surprising that known Picralima alkaloids were not detected as the major constituents of the biologically active dichloromethane extracts, although peaks corresponding to akuammine, .psi.-akuammigine and picraline were observed as minor constituents. The high molecular weight compounds found in the LC-MS of these extracts appeared to be dimers of the previously identified alkaloids, because, in most cases, the observed molecular ion peaks correspond to the expected mass of such dimeric alkaloids.

TABLE 1 ______________________________________ IN VITRO ANTIMALARIAL ACTIVITY OF PICRALIMA NITIDA EXTRACTS AGAINST W-2 AND D-6 CLONES OF PLASMODIUM FALCIPARUM IC.sub.50, .mu.g/ml Extract Plant Part Solvent W-2 Clone D-6 Clone ______________________________________ 1 Seeds CH.sub.2 Cl.sub.2 5.15 5.03 2 Seeds CH.sub.3 OH 7.35 12.99 3 Seeds H.sub.2 O 17.40 12.15 4 Fruit rind CH.sub.2 Cl.sub.2 1.61 2.41 5 Fruit rind CH.sub.3 OH 20.79 32.16 6 Stem bark CH.sub.2 Cl.sub.2 6.46 14.86 7 Seeds Pet. ether 22.81 25.87 8 Seeds CH.sub.3 OH.sup.a 2.25 2.64 9 Stem bark CH.sub.3 OH.sup.a 2.00 1.23 10 Fruit rind CH.sub.3 OH.sup.a 2.16 1.59 ______________________________________ .sup.a Alkaloid fraction

TABLE 2 ______________________________________ THERMOSPRAY LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY (LC-MS) OF THE DICHLOROMETHANE EXTRACT PICRALIMA NITIDA FRUIT RIND. Retention time Peak no. (Min) Major ions M + 1 ______________________________________ 1. 6:12 211(B) 251 2. 11:24 369(B) 383 251 234 210 3. 16:22 410 411 369(B) 368 4. 17:26 339(B) 355 318 5. 22:42 738 808(B) 6. 27:50 349(B) 367 7. 30:40 363 698(B) 261 8. 31:30 367 698 349 9. 43:04 349 435 10. 51:58 685 686(B) 363 318 11. 67:06 757 793 686 435 379(B) 365 349 12. 71:14 685 701(B) 526 463 379 351 349 ______________________________________

TABLE 3 ______________________________________ THERMOSPRAY LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY (LC-MS) OF THE DICHLOROMETHANE EXTRACT OF P. NITIDA SEED Retention time Major ions m/z Peak no. (Min.) (B = base peak) M + 1 ______________________________________ 1. 6:06 274 360? 253 212(B) 198 164 2. 8:34 434 479 390(B) 349 314 299 245 3. 9:34 394 395 354 353(B) 4. 12:08 756 773 738(B) 646 387 370 5. 12:16 752 769 734(B) 408 385 368 6. 14:02 385(B) 386 368 367 355 7. 15:24 388 387 389(B) 355,354 8. 18:24 371 386(B) 354 298 9. 18:42 644 771 386(B) 370 354 298 10. 20:12 357(B) 385 340 300 259 11. 26:22 385 429(B) 370 322 270 12. 28:46 366 649 326(B) 13. 30:14 738 737 414(B) 386 326 14. 33:54 398 413 355(B) 327 15. 37:38 428 444(?) 410 370(B) 355 16. 43:22 413 429 370 369(B) 355 325 17. 51:18 412 413(B) 369 353 18. 59:56 401 467 385 369(B) 325 19. 67:32 678 723 648 467(B) 369 ______________________________________

In the context of this invention, we have tested the major alkaloids of the fruits of P. nitida for in vitro activity against drug resistant and drug sensitive strains of Plasmodium falciparum. The alkaloids showed remarkable inhibitory activity against both clones of P. falciparum at IC.sub.50 values of 0.017-0.9 .mu.g/mL. Among the compounds tested, those belonging to the picraline-akammine subgroup showed the greatest activity, followed by those of the akuammicine type. The alkaloid echitamine showed no activity in this regard.

The structural formulas of the Picralima nitida alkaloids exhibiting in vitro antimalarial activity are as follows: ##STR1##

ANTIMALARIAL IN VITRO BIOASSAY METHOD

The in vitro assays were performed by using a modification of the semi-automated microdilution technique described earlier (Desjardins et al., 1979; Milhous et al., 1985). Two P. falciparum malaria parasite clones, designated as Indochina (W-2) and Sierra Leone (D-6), were utilized in susceptibility testing. The W-2 clone is resistant to chloroquine, pyrimethamine, sulfadoxine, and quinine, and the other clone is resistant to mafloquine.

The test extracts were dissolved in DMSO and serially diluted with media. The uptake of tritiated hypoxanthine was used an an index of inhibition of parasite growth.

TABLE 4 ______________________________________ IN VITRO ANTIMALARIAL ACTIVITY OF PICRALIMA NITIDA EXTRACTS AGAINST W-2 AND D-6 CLONES OF PLASMODIUM FALCIPARUM IC.sub.50, .mu.g/ml Extract Plant Part Solvent W-2 Clone D-6 Clone ______________________________________ 1 Seeds CH.sub.2 Cl.sub.2 5.15 5.03 2 Seeds CH.sub.3 OH 7.35 12.99 3 Seeds H.sub.2 O 17.40 12.15 4 Fruit rind CH.sub.2 Cl.sub.2 1.61 2.41 5 Fruit rind CH.sub.3 OH 20.79 32.16 6 Stem bark CH.sub.2 Cl.sub.2 6.46 14.86 7 Seeds Pet. ether 22.81 25.87 8 Seeds CH.sub.3 OH.sup.a 0.54 0.79 9 Stem bark CH.sub.3 OH.sup.a 2.00 1.23 10 Fruit rind CH.sub.3 OH.sup.a 2.16 1.59 ______________________________________ .sup.a Alkaloid fraction

TABLE 5 ______________________________________ IN VITRO ANTIMALARIAL ACTIVITY OF PICRALIMA NITIDA ALKALOIDS AGAINST W-2 AND D-6 CLONES OF PLASMODIUM FALCIPARUM IC.sub.50, .mu.g/ml COMPOUND W-2 Clone D-6 Clone ______________________________________ Alstonine 0.09 0.02 Alstonine (tetrahydro-) 2.86 2.76 Akuammine 0.66 0.95 .PSI.-Aukuammigine 0.10 0.83 Picraline 0.53 0.78 Akuammicine 0.73 0.45 Echitamine 7.25 4.68 Yohimbine 6.16 7.51 PNF-S7 10.60 7.60 Sarpagine 29.17 16.65 Ajmaline 1.24 4.70 NS-6A 0.003 0.002 Chloroquine 0.04 0.006 Artemisinin 0.002 0.004 Quinine 1.20 0.005 ______________________________________ ##STR2##

EVALUATION OF PLANT EXTRACTS FOR ANTILEISHMANIAL ACTIVITY USING A MECHANISM BASED RADIORESPIRATORY MICROTECHNIQUE (RAM)

Radiorespirometric microtest based on in vitro inhibition of catabolism of .sup.14 CO.sub.2 of a battery of .sup.14 CO.sub.2 substrates by promastigotes, has been used to examine extracts of 11 plants used in Nigerian traditional medicine for possible antileishmanial activity. Of 13 methanol extracts tested, 5 from Gongronema latifolia, Dorstenia multiradiata, Picralima nitida, Cola attiensis, and Desmodium gangeticum, were active at concentrations of 50 .mu.g/ml or less against visceral Leishmania isolate.

INTRODUCTION

Infections due to protozoa of the genus Leishmania 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. The pathological effects of the disease are complex manifesting in various forms, ranging from self-healing cutaneous lesions; recurrent leishmaniasis recidivans; disfiguring mucocutaneous and diffuse cutaneous diseases; to fatal systemic infection, visceral leishmaniasis or kala azar. In the later form, the reticuloendoethelial system is infected with the resultant toll on the spleen, liver, bone marrow, lymph glands, and, often, some degree of intestinal tract dysfunction. Approximately 350 million people within 80 countries are threatened by the disease worldwide.

Clinical drug intervention is presently limited to the use of pentavelent antimonials (SbV), sodium stilbogluconate and N-methylglucamine antimonate, and, secondarily, amphotericin or pentamidine. These antileishmanials require parenteral administration with clinical supervision or hospitalization during treatment because of the severity of possible toxic side-effects that include cardiac and/or renal failure. Treatment with the aforementioned agents is not consistently effective particularly for the most virulent leishmanial disease forms. The World Health Organization has reported large scale resistance of kala azar to SbV, which are the preferred chemotherapy for treatment of most forms of leishmanial disease.sup.36. In some endemic regions, it has been observed that prolonged medication (22 months or more) with SbV is required to effect a clinical cure.sup.37. Long term SbV therapy, however, is not usually advocated due to the mentioned cardiac and renal toxicity of SbV. There is, therefore, a need for the development of more effective, less toxic and orally active antileishmanial agents.

Development of a new drug for the treatment of leishmaniasis has been impeded by the lack of a simple, rapid and universally applicable (to the various Leishmania species/strains infecting humans) drug evaluation system.sup.38. 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.sup.39. Current methods for screening 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.sup.40. 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, thereby making them unsuitable for primary screening of potential antileishmanial agents.

An in vitro radiorespirometric microtest using promastigotes has been developed which relies on drug inhibition of parasite production of .sup.14 CO.sub.2 03 +3* U tery of .sup.14 C-substrates by promastigotes to detect drug-mediated parasite damage at low drug concentration within a short time.sup.41. 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 to patients response to SbV therapy.sup.42.

Visceral leishmaniasis is endemic to the central Nigerian highlands, and zoonotic cutaneous leishmaniasis, prevalent in the northern half of the country. Because of limited supply, expense and toxicity of commercial antileishmanials, traditional herbal therapy is frequently utilized in many leishmanial endemic regions of Nigeria.

In this study, we have used the radiorespirometric microtest (RAM) to evaluate extracts of 11 plants used in Nigerian folk medicine as antiparasitic remedies for possible antileishmanial activity.

MATERIALS AND METHODS

Plant Materials

Plants were selected from a collection made as part of a Salvage Ethnography Project, Institute of African Studies, University of Nigeria Nsukka. Samples were authenticated by Dr. C. O. Okunji of the Department of Pharmacognosy, University of Nigeria Nsukka and Mr. F. Ozioko of the Department of Botany of the same University. Voucher specimens have been deposited in the Pharmacy Herbarium of the University of Nigeria Nsukka.

Extraction Procedure

Two hundred grams of powdered material from each plant was percolated with 80% methanol and concentrated to a sticky gum under reduced pressure. The extracts from the seed materials were pationed between chloroform and water and the two fractions submitted to bioassay. The list of extracts prepared and the laboratory codes are shown in Table 1.

TABLE 6 ______________________________________ Plant Test Species Plant Family Part Solvent Code ______________________________________ Afromomum Zingiberaceae Rhizome MeOH ADF danielli Cola attiensis Sterculiaceae Seed CH.sub.2 Cl.sub.2 CT-1 MeOH CT-2 Crescentia cujeta Bignoniaceae Fruit MeOH CCX Desmodium Fabaceae Leaf MeOH SM gangeticum Dorstenia Moraceae Leaf MeOH DL multiradiata Dracaena mannii Agavaceae Leaf MeOH DM Garcinia kola Guttiferae Seed MeOH GKX Gongronema Asclepiadaceae Leaf MeOH GG latifolia Picralima nitida Apocynaceae Seed CH.sub.2 Cl.sub.2 HB MeOH PN Rothmania Loganiaceae Fruit MeOH RQ withfieldii Schumaniophyton Loganiaceae Leaf MeOH SCM magnificum ______________________________________

Leishmania species/strains

A clinical isolate of visceral Leishmania (Leishmania) chagasi, MHOM/BR/84/BA-13, was used for this study. This isolate was selected because sensitivity to SbV was previously determined using RAM. MHOM/BR/84/BA-13 is sensitive to Pentostam (sodium antimony gluconate) at 6 .mu.g/ml Sb (20 .mu.g/ml drug); and to Glucantime (N-methyl-glucamine antimoniate) at 80 .mu.g/ml Sb (286 .mu.g/ml drug)

Cultivation Medium

Promastigotes of L. chagasi were grown in a serum-free, defined medium, MM2.sup.43. The MM2 medium contained 120 .mu.g/ml protein (10 .mu.g/ml human transferrin, 10 .mu.g/ml human insulin, 100 .mu.g/ml defatted bovine albumin), plus 10 .mu.g/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.sup.44. Cultures were maintained at 25.degree. C. during growth and incubation with drug.

14C-Substrates

The .sup.14 C-labelled substrates and commercial sources are listed in Table 2. For use in radiorespirometry, the .sup.14 C-substrates were diluted to a final concentration of 100,000 disintegrations per minute (dpm)/25 .mu.l using a phosphate buffered balanced salt solution (PBSS: NaCl 6.58 g, KCl 0.4 g, CaCl.sub.2 0.14 g, KH.sub.2 PO.sub.4 0.06 g, MgSO.sub.4 0.05 g, sodium phosphate 0.01 M, made up to 1 l with sterile glass-distilled H.sub.2 O, final pH 7.4). The .sup.14 C-substrates were filter sterilized (0.22 .mu.m Acrodisc filter, Millipore Corporation, Bedford, Mass.) into sterile screwcap vials and stored at 4.degree. C. until use. Subsequent to sterilization, .sup.14 C-substrate vials were opened only within a laminar hood.

Radiorespirometric procedure

Promastigotes were maintained in log phase growth for 3 successive transfers (48-72 hours apart) prior to radiorespirometric testing. Test extracts (or PBSS plus drug solvent [DMSO], for parallel control cultures) was added 24 hours 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.sup.45.

To each well of a microtiter tray (Biospherics Type T010+C010, Universal Plastics & Engineering Company, Rockville, Md.) were added 25 .mu.l of a single .sup.14 C-substrate (100,000 dpm). The tray was covered with a friction-fit lid to prevent evaporation while the promastigotes were being 3.times.centrifugally (700.times.G, 10 min, 4 C) washed free of nutrient medium and drug using PBSS. The final organism pellet was resuspended to a concentration of 1.times.10.sup.9 organisms/ml in PBSS. After the addition of 25 .mu.l of organism suspension to each well (total volume per well, 50 .mu.l .sup.14 C-substrate +promastigote suspension), the wells were immediately covered with a filter paper disc (22mm, #410, Schleicher & Schuell, Inc.,Keene, N.H.) which had been premoistened with one drop of saturated Ba(OH).sub.2 solution. The trays were recovered with the lid. If during the 30 minute incubation at 33.degree. C., the Leishmania metabolize the .sup.14 C-substrates to .sup.14 CO.sub.2, the radioactive gas was collected as a Ba.sup.14 CO.sub.3 precipitate on the filter paper discs. After the incubation, the filter discs were removed from the trays, dried using an infrared lamp, and the .sup.14 C quantity determined using an argon:methane (P10 mixture 9:1 v/v, respectively) gas-flow proportional counter (model 5100, Tennelec, Inc., Oakridge, Tenn.). Data (dmp corrected for background, 1 count per minute; and machine efficiency) were electronically sent to a computer for analysis and graphic presentation.

To obtain a quantitative measure of replicate test variability, tests were initially repeated in duplicate on 4-5 separate days (8-10 tests/drug concentration/organism). The mean dpm/.sup.14 C-substrate had a linear relationship to the magnitude of the standard deviation (SD) in our previous study.sup.46. It was established from the analysis of previous data on the test system that a linear relationship between dpm and SD, existed. Thereafter testing was only repeated in quadruplicate (duplicate tests on two separate days), for each test extract or control compound.

Drug test procedure

The procedure was conducted as described.sup.47 at the extract concentration of .mu.g/ml. A flow chart and diagram of the test method are shown (FIGS. 1 & 2). Drug sensitivity or resistance to SbV drugs was based on .sup.14 C-substrate(s) (Table 1) for which .sup.14 CO.sub.2 release was decreased for drug-treated parasites compared to parallel tests of phosphate buffered balanced salt solution (PBSS+DMSO)-treated (=drug vehicle) controls.

Each expirement consisted of parallel: (a) duplicate tests of drug-treated parasites; plus (b) duplicate tests of drug vehicle control-treated parasites; plus (c) one "nonbiological" sterility control. The nonbiological control consisted of each 14/C-substrate (one substrate per microtiter tray well), and PBSS (the same PBSS batch used to wash, to suspend the parasites, and to make drug solution). Since there were no parasites in the nonbiological control, any 14/CO/2 detected was attributed either to biologic (or, less likely, chemical-) contamination of the 14/C-substrates resulting in breakdown of the 14/C-substrates. If radioactivity above background (=10 disintegration per minute, dpm) was detected in the nonbiological control, the suspect solution(s) was replaced and the experiment was repeated.

______________________________________ PLANTS WITH IN VITRO ANTILEISHMANIAL ACTIVITY Species Plant Part Test Code ______________________________________ 1. Afromomum danielli rhizome ADF 2. Cola attiensis seed CT* 3. Crescentia cujeta fruit CCX 4. Desmodium gangeticum leaf SM* 5. Dorstenia multiradiata leaf DL* 6. Draceana manii leaf DM 7. Garcinia kola seed GKX 8. Gongronema latifolia leaf GG* 9. Picralima nitida fruit HB* 10. Rothmania withfieldii fruit RQ* 11. Schumaniophyton magnificum leaf SCM ______________________________________

TABLE 7 ______________________________________ Numeric code abbreviations of .sup.14 C-substrates used for drug tests* Commercial Numeric Code 14C-Substrates.sup.+ Source ______________________________________ 2 L-Arginine (guanido-14C) A.sup.++ 3 L-Aspartic Acid (4-14C) A 4 L-Asparagine (U-14C) A 5 L-Glutamic Acid (U-14C) A 6 L-Glutamine (U-14C) A 7 Glycine (U-14C) A 9 L-Isoleucine (U-14C) A 10 L-Leucine (1-14C) A 12 L-Methionine (1-14C) A 13 L-Orithine (1-14C) A 15 L-Proline (U-14C) A 17 Taurine (U-14C) A 18 L-Threonine (U-14C) A 20 Tyramine (7-14C) A 24 L-Fucose (1-14C) A 25 D-Galactose (1-14C) A 28 D-Mannose (1-14C) A 42 Orotic Acid (carboxyl-14C) N.sup.ss 44 Succinic Acid (1,4-14C) N 46 Na-n-Butyric Acid (1-14C) A 49 D-Glucosamine (1-14C) A 52 Na-Glycocholic Acid (1-14C) A 53 L-Methionine (methyl-14C) A ______________________________________ *All 14Csubstrates were selected with specific activities as close to 50 mCi/mM/carbon atom as obtainable from commercial sources. .sup.+ A "U" in the 14C designation indicates a "uniform" 14Clabel at eac carbon atom in the molecule. .sup. ++ Amersham, Arlington Heights, IL .sup.ss New England Nuclear, Boston, MA

RESULTS

At a concentration of 50 .mu.g/ml, 5 of the 11 plant extracts tested inhibited the catabolism of two or more of the substrates to CO.sub.2 (Table 3). Cola attiensis extract (CT) inhibited parasite catabolism of 5 of the 21 substrates used in the assay, with the strongest activity observed on the disintegration of ornithine, L-proline, L-aspartic acid (FIG. 1). Gongronema (GG) displayed strong inhibition of the catabolism of succinic acid, D-galactose, D-mannose, L-aspartic acid, L-glutamine and D-glucosamine, as well as L-proline, Na-n-butyric acid, and L-gultamic acid (FIG. 2.). For Picralima extract (HB), the strongest activity was observed against butyric acid, with the drug treated parasite cultures showing a suppression of more than 90% when compared with the values observed for the controls. Strong activity was also recorded for succinic acid, glycine and aspartic acid, and inhibition rate of 40% or more was observed with glutamic acid, glutamine, and methionine (FIGS. 3A-3D). No significant inhibition occurred in the catabolism of tyramine, taurine and fucose at the dose of HB tested.

The extract of Dorstenia (DL) strongly inhibited the catabolism of ornithine, butyric acid, and mannose (FIGS. 4A-4C). Moderate inhibition was observed on aspartic acid, glutamic acid, and threonine. The extract, however, caused an enhancement in the catabolism of fucose, succinic acid, and leucine. Desmodium extract (SM) showed strong inhibition of 5 of the 17 substrates used in the study, with the strongest inhibition observed against arginine and L-fucose.

Diseases due to protozoal infections are largely a problem of developing countries. Because of the unavailability of effective and affordable drugs, many of the people in the leishmaniases endemic areas rely on tradidional systems of medicine for treatment. Scientific evaluation of medicinal plants used in the preparation of such traditional remedies are useful in the search for more effective and less toxic therapeutic agents. Plants used for this study were selected from a list of plants used in traditional medicine in Nigeria for the treatment of parasitic infections. Nigeria has an extensive history of successful treatment of native leishmanial and other protozoan diseases using traditional medicines from native plants. Nigerial antiparasitic plant extracts are locally available, inexpensive, administered orally, and have a long precedent of human use because of effectiveness and low adverse reaction.

The results show that the extracts could be explored as sources of leads for new antileishmanial agents. The extracts displayed varied inhibition patterns which suggests different mechanisms in their mode of action.

Two of the extracts, CT and DL appear to be more active against amino acid catabolism, whereas HB, SM and GG showed preferential inhibition against sugars and fatty acids.

One of the plants investigated, Cola attiensis is used, among other things, for the treatment of migraine, bronchitis, and catarrh. Picralima nitida has been employed in the treatment of malaria, African sleeping sickness, and bacterial infection. Desmodium gangeticumis 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 Dorstenia multiradiata is used as an antiviral agent as as a local anti-inflammatory Gongronema is valued as a bitter tonic, and the alcoholic infusion is dispensed for bilharzia, viral hepatitis and as a general antimicrobial agent.

Pentavelent antimonials have a serum half-life of 2 hours with the maximum achievable serum level of approximately 20 .mu.g/ml Sb (or approximately 73 .mu.g/ml drug).sup.48. It is interesting to note that even as crude mixtures, the 5 active plant extracts (Table 3, FIGS. 1-5) were active at 50 .mu.g/ml and one, DL-55, retained antileishmanial activity to 5 .mu.g/ml. Crude extract antileishmanial activity, at drug concentrations comparable to SbV, seems to indicate high potential fo the active drug principles as a new antileishmanials.

The plants are presently being analyzed for their chemical constituents. Literature, however, revealed that the plants vary in their constituents. P. nitida contains indole alkaloids as the major components.sup.49, D. gangeticum yields .beta.-carbolines and phenylethylamines.sup.50. There is no available report on any previous chemical analysis of Cola attiensis, Gonoronema latifolia, or Dorstenia multiradiata.

NOVEL ANTILEISHMANIAL INDOLE ALKALOIDS FROM FRUITS OF PICRALIMA NITIDA

Methanol and aqueous extracts of the West African tree Picralima nitida showed significant inhibition of leishmania promastigotes. Using a radiorespirometric microtest based on in vitro inhibition of catabolism of 14/CO/2 of a battery of 14/C-substrates by promastigotes, the extracts were found active at concentrations of 50 .mu.g/ml or less against visceral Leishmania chagasi and cutaneous L. mexicana. The extracts significantly inhibited (10%-90%) the catabolism of certain sugars, amino acids, or fatty acid precursors by promastigotes. The indole alkaloids akuammine, pseudo-akuammigine, picraline, alstonine and akuammicine were isolated from the active fraction. The greatest inhibition was observed with alstonine. The compound a dose related activity with the highest growth inhibition observed at 50 .mu.g/ml. At 20 .mu.g/ml the compound showed a growth of 69.3% after 96 hours.

______________________________________ Leishmania (Leishmania) chagasi, MHOM/BR/84/BA-13, MM2 medium, 96 hrs HB-1 Plant Extract (20 .mu.g/ml), Orig. File: 910625HB, 10-9 pros/ml, DMSO final concentration 0.58% CONTROL TEST .sup.14 C-SUB- MEAN CONTROL MEAN TEST STRATES n = 8 SDEV n = 3 SDEV ______________________________________ L-Aspartic 9,322 2,318 18,972 3,593 Acid (4-.sup.14 C) L-Glutamine 1,519 260 2,771 834 (U-.sup.14 C) L-Glycine 209 98 163 130 (U-.sup.14 C) L-Ornithine 698 162 1,084 53 (1-.sup.14 C) Succinic Acid 330 67 216 58 (1,4-.sup.14 C) Na-n-Butyric 1,172 225 406 35 Acid (a-.sup.14 C) ______________________________________ NOTE: Growth inhibition over 96 hours was 69.3%: Control cells were 3.12 .times 10.sup.7 pros/ml (624 .times. 50,000), whereas HB1-treated were 0.96 .times. 10.sup.7 pros/ml (192 .times. 50,000, 30.7% Control). Pentavelent antimonials do not produce visible growth inhibition at 20 .mu.g/ml Sb (7 .mu.g/ml drug)

______________________________________ Leishmania (Leishmania) chagasi, MHOM/BR/84/BA-13, MM2 medium, 96 hrs HB-1 Plant Extract (10 .mu.g/ml), Orig. File: 910619HB, 10-9 pros/ml, DMSO final concentration 0.58% CONTROL TEST .sup.14 C-SUB- MEAN CONTROL MEAN TEST STRATES n = 4 SDEV n = 4 SDEV ______________________________________ L-Aspartic 24,695 7,078 24,884 3,703 Acid (4-.sup.14 C) L-Glutamine 6,316 718 8,069 405 (U-.sup.14 C) L-Glycine 587 47 536 61 (U-.sup.14 C) L-Ornithine 3,206 433 5,129 543 (1-.sup.14 C) Succinic Acid 313 32 344 87 (1,4-.sup.14 C) Na-n-Butyric 3,599 149 5,080 272 Acid (a-.sup.14 C) ______________________________________

______________________________________ Leishmania (Leishmania) chagasi, MHOM/BR/84/BA-13, MM2 medium, 96 hrs HB-1 Plant Extract (10 .mu.g/ml), Orig. File: 910628HB, 10-9 pros/ml, DMSO final concentration 0.58% CONTROL TEST .sup.14 C-SUB- MEAN CONTROL MEAN TEST STRATES n = 8 SDEV n = 8 SDEV ______________________________________ L-Aspartic 11,544 3,274 12,851 1,092 Acid (4-.sup.14 C) L-Glutamine 2,524 533 5,084 956 (U-.sup.14 C) L-Glycine 177 21 226 24 (U-.sup.14 C) L-Ornithine 1,282 281 3,194 400 (1-.sup.14 C) Succinic Acid 280 51 640 105 (1,4-.sup.14 C) Na-n-Butyric 2,021 571 3,296 1,256 Acid (a-.sup.14 C) ______________________________________

______________________________________ Leishmania (Leishmania) chagasi, MHOM/BR/84/BA-13, MM2 medium, 96 hrs HB-1 Plant Extract (1 .mu.g/ml), Orig. File: 910618HB, 10-9 pros/ml, DMSO final concentration 0.58% CONTROL TEST .sup.14 C-SUB- MEAN CONTROL MEAN TEST STRATES n = 8 SDEV n = 3 SDEV ______________________________________ L-Aspartic 32,733 3,503 31,073 1,693 Acid (4-.sup.14 C) L-Glutamine 12,389 1,932 12,453 1,210 (U-.sup.14 C) L-Glycine 681 157 478 49 (U-.sup.14 C) L-Ornithine 10,399 3,090 10,560 706 (1-.sup.14 C) Succinic Acid 2,550 344 1,377 367 (1,4-.sup.14 C) Na-n-Butyric 5,739 755 6,331 398 Acid (a-.sup.14 C) ______________________________________

NEW LEADS TO THE TREATMENT OF TRYPANOSOMIASIS BASED ON ISOLATES FROM PLANTS USED IN TRADITIONAL MEDICINE

Available chemotherapeutiv agents for the treatment of trypanosomiases are generally unstaisfactory, as most of the drugs are very toxic and cases of druc resistance are becoming widespread. We have examined extracts of twleve plants used in traditional medicine in South-Eastern Nigeria antiparasitic agents for possible antitrypanosomial activity.

From the in vivo inhibition of the development of Trypanosoma brucei brucei in mice and rats, extracts of two of the species, Picralima nitada, and Dorstenia multiradiata were found active at very low doses.

An intraperitoneal dose of 50 mg/kg and 5 mg/kg of the methanol and aqueous extracts respectively of Picralima seeds completely cleared animals of the parasites at post-treatment day 12 in rats and day 10 in the mouse model. Methanol extract of Dorstenia gave similar results at treatment day 10 and 8 for the rat and mouse models respectively.

The indole alkaloids akuammine, pseudo-akuammigine, picraline, alstonine and akuammicine were isolated from the active fraction of P. nitida, whereas anthocyanidins were the active components of the extract from D. multiradiata.

______________________________________ IN VIVO ANTITRYPANOSOMIAL ACTIVITY OF PICRALIMA NITIDA EXTRACTS Animal Day of 0% Test Substance Dose Model Parasite Count ______________________________________ MeOH Extract 50 mg rat 12 MeOH Extract 50 mg mouse 10 H.sub.2 O Extract 5 mg rat 12 H.sub.2 O Extract 5 mg mouse 10 Berenil 7 mg rat 8 Berenil 7 mg mouse 6 ______________________________________ *Dosing by i.p. route *Paarasitemia was detected on day 21 after treatment