Abstract:
A method of preparing substantially purified alkaloids from seeds, stems, uit-rind and bark of a plant selected from Picralima nitida, Gongronema latifolia, Dorstenia multiradiata, Cola attiensis, Rothmania withfieldii and Desmodium gangeticum, for use in the treatment of protozoal diseases, comprising: 
     pulverizing said plant; 
     a first solvent, drying the extracted material and re-extracting the dried material with a different solvent; 
     extracting a fresh sample of said plant with boiling water; 
     filtering and concentrating the boiling water solvent extracts under reduced pressure; 
     concentrating the dried extract to a gum and re-extracting said gum with an aqueous acidic HCl solution; 
     filtering the acidic extract and making it alkaline to a pH of about 9 with a concentration NaOH solution; 
     extracting the alkaline solution with dichloromethane; 
     concentrating organic layers of the extracted alkaline solution to dryness under reduced pressure to obtain an alkaloid fraction; and 
     separating the alkaloid fraction by liquid chromotography-mass spectrometry to obtain substantially purified alkaloids for use in treatment of protozoal diseases.

Description:
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 50  values of 0.0.1-0.9 μg/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 &#34;Akuamma&#34; seeds, employed extensively in west Africa as an ingredient in many folk remedies 1 , 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 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 4 , 5 . The principal alkaloid found in the plant, akuammine, has been shown to be inactive against avian malaria and in clinical trials 6 . 
    
    
    
     Akuammine, however, is a strong sympathicomimetic and possesses local anesthetic action comparable to that of cocaine 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 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 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 -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 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 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 12 , 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 14 . 
    
    
    
     Treatment with the aforementioned agents is not consistently effective particularly for the most virulent leishmanial disease forms 15 , 16 , 17 , 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 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 20 , 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 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 &#34;insensitive&#34; within in vitro assays to SbV compounds used for human leishmaniases 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 24 , 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  14  CO 2  03 +3* U  W  Etery of  14  C-substrates by promastigotes to detect drug-mediated parasite damage at low drug concentration within a short time 26 , 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 &#34;difficult to grow&#34; species. The method has been shown to correlate to patients response to SbV therapy 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 μg/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 μg/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 μg/ml. At 20 μg/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  14  CO 2  of a battery of  14  CO 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  14  CO 2  of a battery of  14  CO 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 2  Cl 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°-60°) extraction of the seeds. The fruit rind (200 g) and the stem bark (100 g) were similarly extracted with CH 2  Cl 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 10×200 ml CH 2  Cl 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 ψ-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. 29  and Milhous et al. 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 18  columns were eluted with CH 3  CN--H 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° C. with the thermospray probe run at T 1  =105° C. and T 2  =190° 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 50  values ranging from 1.23 μg/mL to 32.16 μg/mL. The CH 2  Cl 2  extracts (1,4,6) showed the strongest activity when compared to the methanolic (2,5) and aqueous (3) extracts with IC 50  of 1.61 μg/ml and 5.15 μg/ml for the fruit rind and seeds, respectively, in the W-2 plasmodium clone; and 5.03 μg/ml and 2.4 μg/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 2  Cl 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 2  Cl 2  extracts of either the fruit rind or seeds of P. nitida. Akuammine (M+1=m/z 383), ψ-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 (&gt;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 31  ; Khalid et al. 32  ; Cubukcu et al. 33  The activity of these extracts are apparently weaker (from the relative IC 50  values) than those of the clinically useful antimalarials of plant origin, quinine (cf. Warhurst 34 ) and artemisinin (cf. Klayman 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 50  value of the stem bark was reduced from 6.46 μg/mL for the crude extract to 2.25 μg/mL in the Draggendorf positive fraction when tested against the W-2 clone, and in the D-6 model, the IC 50  values of 14.86 μg/mL and 1.23 μg/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, ψ-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 OFPICRALIMA NITIDA EXTRACTS AGAINST W-2AND D-6 CLONES OF PLASMODIUM FALCIPARUM              IC.sub.50, μg/mlExtract  Plant Part Solvent    W-2 Clone                                D-6 Clone______________________________________1      Seeds      CH.sub.2 Cl.sub.2                        5.15     5.032      Seeds      CH.sub.3 OH                        7.35    12.993      Seeds      H.sub.2 O  17.40   12.154      Fruit rind CH.sub.2 Cl.sub.2                        1.61     2.415      Fruit rind CH.sub.3 OH                        20.79   32.166      Stem bark  CH.sub.2 Cl.sub.2                        6.46    14.867      Seeds      Pet. ether 22.81   25.878      Seeds      CH.sub.3 OH.sup.a                        2.25     2.649      Stem bark  CH.sub.3 OH.sup.a                        2.00     1.2310     Fruit rind CH.sub.3 OH.sup.a                        2.16     1.59______________________________________ .sup.a Alkaloid fraction 
    
     
                       TABLE 2______________________________________THERMOSPRAY LIQUID CHROMATOGRAPHY-MASSSPECTROMETRY (LC-MS) OF THEDICHLOROMETHANE EXTRACTPICRALIMA NITIDA FRUIT RIND.    Retention timePeak no. (Min)          Major ions M + 1______________________________________1.        6:12          211(B)     2512.       11:24          369(B)     383                   251                   234                   2103.       16:22          410        411                   369(B)                   3684.       17:26          339(B)     355                   3185.       22:42          738        808(B)6.       27:50          349(B)     3677.       30:40          363        698(B)                   2618.       31:30          367        698                   3499.       43:04          349        43510.      51:58          685        686(B)                   363                   31811.      67:06          757        793                   686                   435                   379(B)                   365                   34912.      71:14          685        701(B)                   526                   463                   379                   351                   349______________________________________ 
    
     
                       TABLE 3______________________________________THERMOSPRAY LIQUID CHROMATOGRAPHY-MASSSPECTROMETRY (LC-MS) OF THEDICHLOROMETHANE EXTRACT OF P. NITIDA SEED    Retention time                  Major ions m/zPeak 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                  29810.      20:12         357(B)        385                  340                  300                  25911.      26:22         385           429(B)                  370                  322                  27012.      28:46         366           649                  326(B)13.      30:14         738           737                  414(B)                  386                  32614.      33:54         398           413                  355(B)                  32715.      37:38         428           444(?)                  410                  370(B)                  35516.      43:22         413           429                  370                  369(B)                  355                  32517.      51:18         412           413(B)                  369                  35318.      59:56         401           467                  385                  369(B)                  32519.      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 50  values of 0.017-0.9 μg/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 OFPICRALIMA NITIDA EXTRACTS AGAINST W-2 AND D-6CLONES OF PLASMODIUM FALCIPARUM              IC.sub.50, μg/mlExtract  Plant Part Solvent    W-2 Clone                                D-6 Clone______________________________________1      Seeds      CH.sub.2 Cl.sub.2                        5.15     5.032      Seeds      CH.sub.3 OH                        7.35    12.993      Seeds      H.sub.2 O  17.40   12.154      Fruit rind CH.sub.2 Cl.sub.2                        1.61     2.415      Fruit rind CH.sub.3 OH                        20.79   32.166      Stem bark  CH.sub.2 Cl.sub.2                        6.46    14.867      Seeds      Pet. ether 22.81   25.878      Seeds      CH.sub.3 OH.sup.a                        0.54     0.799      Stem bark  CH.sub.3 OH.sup.a                        2.00     1.2310     Fruit rind CH.sub.3 OH.sup.a                        2.16     1.59______________________________________ .sup.a Alkaloid fraction 
    
     
                       TABLE 5______________________________________IN VITRO ANTIMALARIAL ACTIVITY OFPICRALIMA NITIDA ALKALOIDS AGAINSTW-2 AND D-6 CLONES OF PLASMODIUM FALCIPARUM           IC.sub.50, μg/mlCOMPOUND          W-2 Clone D-6 Clone______________________________________Alstonine         0.09      0.02Alstonine (tetrahydro-)             2.86      2.76Akuammine         0.66      0.95Ψ-Aukuammigine             0.10      0.83Picraline         0.53      0.78Akuammicine       0.73      0.45Echitamine        7.25      4.68Yohimbine         6.16      7.51PNF-S7            10.60     7.60Sarpagine         29.17     16.65Ajmaline          1.24      4.70NS-6A              0.003     0.002Chloroquine       0.04       0.006Artemisinin        0.002     0.004Quinine           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  14  CO 2  of a battery of  14  CO 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 μg/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 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 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 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 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 &#34;insensitive&#34; within in vitro assays to SbV compounds used for human leishmaniases 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  14  CO 2  03 +3* U   tery of  14  C-substrates by promastigotes to detect drug-mediated parasite damage at low drug concentration within a short time 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 &#34;difficult to grow&#34; species. The method has been shown to correlate to patients response to SbV therapy 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           TestSpecies    Plant Family                  Part     Solvent                                  Code______________________________________Afromomum  Zingiberaceae                  Rhizome  MeOH   ADFdanielliCola attiensis      Sterculiaceae                  Seed     CH.sub.2 Cl.sub.2                                  CT-1                           MeOH   CT-2Crescentia cujeta      Bignoniaceae                  Fruit    MeOH   CCXDesmodium  Fabaceae    Leaf     MeOH   SMgangeticumDorstenia  Moraceae    Leaf     MeOH   DLmultiradiataDracaena mannii      Agavaceae   Leaf     MeOH   DMGarcinia kola      Guttiferae  Seed     MeOH   GKXGongronema Asclepiadaceae                  Leaf     MeOH   GGlatifoliaPicralima nitida      Apocynaceae Seed     CH.sub.2 Cl.sub.2                                  HB                           MeOH   PNRothmania  Loganiaceae Fruit    MeOH   RQwithfieldiiSchumaniophyton      Loganiaceae Leaf     MeOH   SCMmagnificum______________________________________ 
    
     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 μg/ml Sb (20 μg/ml drug); and to Glucantime (N-methyl-glucamine antimoniate) at 80 μg/ml Sb (286 μg/ml drug) 
     Cultivation Medium 
     Promastigotes of L. chagasi were grown in a serum-free, defined medium, MM2 43 . The MM2 medium contained 120 μg/ml protein (10 μg/ml human transferrin, 10 μg/ml human insulin, 100 μg/ml defatted bovine albumin), plus 10 μg/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 44 . Cultures were maintained at 25° C. during growth and incubation with drug. 
    
    
     14C-Substrates 
     The  14  C-labelled substrates and commercial sources are listed in Table 2. For use in radiorespirometry, the  14  C-substrates were diluted to a final concentration of 100,000 disintegrations per minute (dpm)/25 μl using a phosphate buffered balanced salt solution (PBSS: NaCl 6.58 g, KCl 0.4 g, CaCl 2  0.14  g, KH 2  PO 4  0.06 g, MgSO 4  0.05 g, sodium phosphate 0.01 M, made up to 1 l with sterile glass-distilled H 2  O, final pH 7.4). The  14  C-substrates were filter sterilized (0.22 μm Acrodisc filter, Millipore Corporation, Bedford, Mass.) into sterile screwcap vials and stored at 4° C. until use. Subsequent to sterilization,  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 45 . 
    
     To each well of a microtiter tray (Biospherics Type T010+C010, Universal Plastics &amp; Engineering Company, Rockville, Md.) were added 25 μl of a single  14  C-substrate (100,000 dpm). The tray was covered with a friction-fit lid to prevent evaporation while the promastigotes were being 3×centrifugally (700×G, 10 min, 4 C) washed free of nutrient medium and drug using PBSS. The final organism pellet was resuspended to a concentration of 1×10 9  organisms/ml in PBSS. After the addition of 25 μl of organism suspension to each well (total volume per well, 50 μl  14  C-substrate +promastigote suspension), the wells were immediately covered with a filter paper disc (22mm, #410, Schleicher &amp; Schuell, Inc.,Keene, N.H.) which had been premoistened with one drop of saturated Ba(OH) 2  solution. The trays were recovered with the lid. If during the 30 minute incubation at 33° C., the Leishmania metabolize the  14  C-substrates to  14  CO 2 , the radioactive gas was collected as a Ba 14  CO 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  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/ 14  C-substrate had a linear relationship to the magnitude of the standard deviation (SD) in our previous study 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 47  at the extract concentration of μg/ml. A flow chart and diagram of the test method are shown (FIGS. 1 &amp; 2). Drug sensitivity or resistance to SbV drugs was based on  14  C-substrate(s) (Table 1) for which  14  CO 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 &#34;nonbiological&#34; 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 ACTIVITYSpecies             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*                        CommercialNumeric 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)                        A10        L-Leucine (1-14C)  A12        L-Methionine (1-14C)                        A13        L-Orithine (1-14C) A15        L-Proline (U-14C)  A17        Taurine (U-14C)    A18        L-Threonine (U-14C)                        A20        Tyramine (7-14C)   A24        L-Fucose (1-14C)   A25        D-Galactose (1-14C)                        A28        D-Mannose (1-14C)  A42        Orotic Acid (carboxyl-14C)                        N.sup.ss44        Succinic Acid (1,4-14C)                        N46        Na-n-Butyric Acid (1-14C)                        A49        D-Glucosamine (1-14C)                        A52        Na-Glycocholic Acid (1-14C)                        A53        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 &#34;U&#34; in the 14C designation indicates a &#34;uniform&#34; 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 μg/ml, 5 of the 11 plant extracts tested inhibited the catabolism of two or more of the substrates to CO 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 μg/ml Sb (or approximately 73 μg/ml drug) 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 μg/ml and one, DL-55, retained antileishmanial activity to 5 μg/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 49 , D. gangeticum yields β-carbolines and phenylethylamines 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 μg/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 μg/ml. At 20 μg/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 μg/ml),Orig. File: 910625HB, 10-9 pros/ml,DMSO final concentration 0.58%    CONTROL               TEST.sup.14 C-SUB-    MEAN       CONTROL    MEAN   TESTSTRATES  n = 8      SDEV       n = 3  SDEV______________________________________L-Aspartic    9,322      2,318      18,972 3,593Acid (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    35Acid (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 × 50,000), whereas HB1-treated were 0.96 × 10.sup.7 pros/ml (192 × 50,000, 30.7% Control). Pentavelent antimonials do not produce visible growth inhibition at 20 μg/ml Sb (7 μg/ml drug) 
    
     
         ______________________________________Leishmania (Leishmania) chagasi, MHOM/BR/84/BA-13,MM2 medium, 96 hrs HB-1 Plant Extract (10 μg/ml),Orig. File: 910619HB, 10-9 pros/ml,DMSO final concentration 0.58%    CONTROL               TEST.sup.14 C-SUB-    MEAN       CONTROL    MEAN   TESTSTRATES  n = 4      SDEV       n = 4  SDEV______________________________________L-Aspartic    24,695     7,078      24,884 3,703Acid (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  272Acid (a-.sup.14 C)______________________________________ 
    
     
         ______________________________________Leishmania (Leishmania) chagasi, MHOM/BR/84/BA-13,MM2 medium, 96 hrs HB-1 Plant Extract (10 μg/ml),Orig. File: 910628HB, 10-9 pros/ml,DMSO final concentration 0.58%    CONTROL               TEST.sup.14 C-SUB-    MEAN       CONTROL    MEAN   TESTSTRATES  n = 8      SDEV       n = 8  SDEV______________________________________L-Aspartic    11,544     3,274      12,851 1,092Acid (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  1,256Acid (a-.sup.14 C)______________________________________ 
    
     
         ______________________________________Leishmania (Leishmania) chagasi, MHOM/BR/84/BA-13,MM2 medium, 96 hrs HB-1 Plant Extract (1 μg/ml),Orig. File: 910618HB, 10-9 pros/ml,DMSO final concentration 0.58%    CONTROL               TEST.sup.14 C-SUB-    MEAN       CONTROL    MEAN   TESTSTRATES  n = 8      SDEV       n = 3  SDEV______________________________________L-Aspartic    32,733     3,503      31,073 1,693Acid (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 398Acid (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 ACTIVITYOF PICRALIMA NITIDA EXTRACTS                 Animal  Day of 0%Test Substance       Dose      Model   Parasite Count______________________________________MeOH Extract       50 mg     rat     12MeOH Extract       50 mg     mouse   10H.sub.2 O Extract       5 mg      rat     12H.sub.2 O Extract       5 mg      mouse   10Berenil     7 mg      rat      8Berenil     7 mg      mouse    6______________________________________ *Dosing by i.p. route *Paarasitemia was detected on day 21 after treatment