Two novel derivatives of clofazimine [3‐(p‐chloroanilino)‐10‐(p‐chlorophenyl)‐2,10‐dihydro‐2‐isopropylimino)phenazine] and the tetramethylpiperidine (TMP)‐substituted phenazines, B4119 [3‐(3‐chloro‐4‐fluoroanilino)‐10‐(3‐chloro‐4‐fluorophenyl)‐2,10‐dihydro‐2(2,2,6,6‐tetramethylpiper‐4‐ylimino)phenazine] and B4158 [3‐(4‐isopropylanilino)‐10‐(4‐isopropylphenyl)‐2,10‐dihydro‐2‐(2,2,6,6‐tetramethylpiper‐4‐ylimino)phenazine] (1–8 μM), were evaluated for activity against chloroquin‐, quinine‐, and sulfadoxine/pyrimethamine‐sensitive and ‐resistant strains of Plasmodium falciparum in vitro, as well as for their effects on polymerisation of haeme to β‐hematin. By using microscopic and flow cytometric methods, it was found that B4119 and B4158, but not clofazimine, inhibited the growth of sensitive, as well as resistant strains of P. falciparum with IC50 values between 0.22 and 0.7 μM, indicating lack of cross‐resistance. Augmentation of anti‐plasmodial activity was observed when B4119 and B4158 were used in combination with chloroquin or mefloquine. Inhibition of the growth of P. falciparum was associated with interference with haeme polymerisation to β‐hematin in vitro, while administration of B4119 to P. berghei‐infected mice was accompanied by a significant reduction in parasitemia and improved therapeutic activity was observed when this agent was combined with chloroquin. The data presented in this study demonstrate that the TMP‐substitution at position 2 on the phenazine nucleus of riminophenazines confers anti‐plasmodial activity on these compounds. These may prove to be useful forerunners in the design of novel anti‐plasmodial pharmacologic agents. Drug Dev. Res. 50:195–202, 2000. © 2000 Wiley‐Liss, Inc.

BackgroundTuberculosis remains a major health problem and knowledge of the diversity of Mycobacterium tuberculosis strains in specific geographical regions can contribute to the control of the disease. This study describes the genetic profile of M. tuberculosis in five districts of Limpopo Province.MethodsA total 487 isolates were collected from the National Health Laboratory Services from all regions/districts of Limpopo Province. Only 215 isolates were confirmed to be M. tuberculosis by Bactec Mycobacterium Growth Indicator Tube 960® and Rhodamine-Auramine staining. Isolates were subcultured on Löwenstein-Jensen medium agar slants to validate purity. They were spoligotyped and data analysed using the international spoligotyping database 4 (SpolDB4).ResultsOf the 215 isolates, 134 (62.3%) were genotyped into 21 genotype families while 81 (37.7%) were orphans. The 81 orphans were further subjected to resolution employing SpolDB3/RIM. Overall, the study revealed a high diversity of strains of 32 predominantly the non-Beijing lineages: the LAM- LAM3 (9.8%), LAM9 (4.7%) and LAM11- ZWE (3.3%), the T-T1(15.0%), T2 (0.9%), T2-T3 (1.4%), the CAS-CAS1-Delhi 5 (1.9%) and CAS1-KILI (1.4%) the MANU2 (1.4%), U (0.5%), X-X1(1.4%), X3 (1.9%), S (9.8%), CAS (1.4%), LAM7(0.9%), T3(0.5%), LAM8(4.7%), T4(1.4%), X2(0.4%), AI5(1.9%), LAM1(0.5%), FAMILY33 (1.9%), EAI4(1.4%), M. microti (1.9%). The Beijing and Beijing-like families were (14.9%) and (0.9%), respectively. A total of 28(13%) clusters and 77(36%) unique cases were identified. Beijing strain (SIT 1) formed the biggest cluster constituting 14%, followed by LAM3 (SIT 33), T1 (SIT 53) and LAM4 (SIT 811) with 7%, 5.1% and 2.8%, respectively. The Beijing family was the only genotype found in all the five districts and was predominant in Mopani (18.8%), Sekhukhune (23.7%) and Vhembe (23.3%). Dominant genotypes in Capricorn and Waterberg were LAM3 (11.9%) and T1 (13.3%), respectively.ConclusionA wide diversity of lineages was demonstrated at district level. A high number of clusters per district provided evidence of on-going transmission in this Province.

Two novel derivatives of clofazimine [3-(p-chloroanilino)-10-(p-chlorophenyl)-2,10-dihydro-2-isopropylimino)phenazine] and the tetramethylpiperidine (TMP)-substituted phenazines, B4119 [3-(3-chloro-4-fluoroanilino)-10-(3-chloro-4-fluorophenyl)-2,10-dihydro-2(2,2,6,6-tetramethylpiper-4ylimino)phenazine] and B4158 [3-(4-isopropylanilino)-10-(4-isopropylphenyl)-2,10-dihydro-2-(2,2,6,6tetramethylpiper-4-ylimino)phenazine] (1-8 µM), were evaluated for activity against chloroquin-, quinine-, and sulfadoxine/pyrimethamine-sensitive and -resistant strains of Plasmodium falciparum in vitro, as well as for their effects on polymerisation of haeme to β-hematin. By using microscopic and flow cytometric methods, it was found that B4119 and B4158, but not clofazimine, inhibited the growth of sensitive, as well as resistant strains of P. falciparum with IC 50 values between 0.22 and 0.7 µM, indicating lack of crossresistance. Augmentation of anti-plasmodial activity was observed when B4119 and B4158 were used in combination with chloroquin or mefloquine. Inhibition of the growth of P. falciparum was associated with interference with haeme polymerisation to β-hematin in vitro, while administration of B4119 to P. bergheiinfected mice was accompanied by a significant reduction in parasitemia and improved therapeutic activity was observed when this agent was combined with chloroquin. The data presented in this study demonstrate that the TMP-substitution at position 2 on the phenazine nucleus of riminophenazines confers antiplasmodial activity on these compounds. These may prove to be useful forerunners in the design of novel anti-plasmodial pharmacologic agents. Drug Dev.

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Background. Rubia cordifolia has been used to treat various diseases and exhibits antimicrobial, anti-inflammatory, analgesic and antidiarrheal activities. The plant has not been investigated for its anti-mycobacterial activity against virulent tuberculosis strains as well as cytotoxic and anti-oxidant activities in mononuclear cells and neutrophils. Methods. Ethanolic and dichloromethane leaf extract fractions of Rubia cordifolia (0.2-125µg/ml) were screened for anti-mycobacterial activity using a fluorescent microplate assay. The anti-oxidant activity of the fractions was tested using the radical scavenging assay, while their cytotoxicity to lymphocytes was measured using the WST-8 assay. Reactive oxygen species production by neutrophils exposed to fractions was tested using Cell Meter™ Fluorimetric ROS Assay kit. Results. The ethanolic leaf fractions exhibited overall superior anti-mycobacterial activity than the dichloromethane group and were further screened for anti-oxidant and cytotoxic activities. Ethanolic sub-fractions K2F-3.2, K2F-3.3 and K2F-3.4 showed significant antioxidant activities at concentration of 50 µg/ml to 200 µg/ml and higher in both the cell free-based radical scavenging activity and total reactive oxygen species production assays. For lymphocytes, the test agents showed anti-proliferative activity at 25 µg/ml to 200 µg/ml for sub-fraction K2F-3.3B and 50 µg/ml to 200 µg/ml for agents K2F-3.2 and K2F-3.4. Conclusion. The current study is the first to record the in vitro anti-mycobacterial, anti-oxidant and cytotoxic activities of Rubia cordifolia plant leaf extract sub-fractions using the given experimental setups and further research activities to identify the bioactive components are to be pursued.