Mitochondria-dependent apoptogenic activity of the aqueous root extract of Croton membranaceus against human BPH-1 cells.

Croton membranaceus aqueous root extract (CMARE) is among the widely used phytotherapeutics in Ghana for the management of benign prostatic hyperplasia (BPH) and prostate cancer. However, the mechanism of action of CMARE remains to be elucidated. This study aimed to establish whether apoptosis is involved in the antiproliferative effect of CMARE on human BPH-1 cells. We determined the effect of treatment with 0, 1, 3, and 5 mg/mL CMARE for 24, 48, and 72 h on the viability and morphology of BPH-1 cells using the MMT assay and phase-contrast microscopy, respectively. We examined the apoptosis-inducing effects of CMARE after 48 h at the cellular level using Hoescht 33258 and JC-1 dye staining and flow cytometry analysis. We performed reverse transcription polymerase chain reaction and Western blotting to confirm the apoptotic effects of CMARE at the molecular level. CMARE induced a significant dose-dependent inhibition in the proliferation of BPH-1 cells (P < 0.05) and an alteration in their morphology and a reduction their density. Furthermore, CMARE induced dose-dependent staining of the nuclear chromatin, significant DNA fragmentation with G0/G1 sub-diploid cells (P < 0.01), and loss of the mitochondrial membrane potential in the treated cells compared to the controls after 48 h (P < 0.01). Additionally, while CMARE induced a significant upregulation of the mRNA and protein levels of Bax, those of Bcl2 did not change significantly. Therefore, induction of mitochondria-dependent apoptosis of BPH-1 cells may be a possible mechanism of action of CMARE.

Treatment of benign prostatic hyperplasia with Croton membranaceus in an experimental animal model.

ETHNOPHARMACOLOGICAL RELEVANCE: Croton membranaceus leaf extracts are used in the Bahamas to aromatize tobacco. In Nigeria it is used to improve digestion and in Ghana, the root extract is used for the treatment of benign prostatic hyperplasia (BPH). Despite claims of efficacy no data exists to support this. The aim of this study was to determine if Croton membranaceus aqueous root extract (CMARE) could attenuate the development of BPH in an animal model. MATERIALS AND METHODS: Fifty (50) adult male Sprague-Dawley rats weighing 200-250g were randomly divided into 5 groups. Group 1 served as the control and received normal saline p.o. Groups 2-5 were castrated and injected with 5mg/kg b.wt. testosterone propionate subcutaneously for 28 days. Group 2 (model group) had no further treatment. Group 3 was simultaneously given 0.5mg/kg b.wt. finasteride p.o. throughout. Groups 4 and 5 received 30mg/kg b.wt. [low dose (LD)] and 300mg/kg b.wt. [high dose (HD)] CMARE, respectively, for 28 days. Rats were sacrificed at the end of the study and all prostate organs harvested. Wet weights, volumes and prostatic index (PI) were determined. Tissues were histologically examined. Serum prostate specific antigen (PSA) and dihydrotestosterone (DHT) levels were determined. RESULTS: Prostate volume of the control group was 0.67±0.23cm(3). The model, finasteride, CMARE LD and HD groups had the following volumes: 0.92±0.12, 0.84±0.16, 0.79±0.16 and 0.80±0.19cm(3), respectively. Only the model group showed significant statistical differences with the control (p=0.007). PI for control, model, finasteride, LD and HD groups was as follows: 0.19±0.04, 0.30±0.04, 0.25±0.04, 0.21±0.05 and 0.22±0.05. No statistical differences between the control PI and the CMARE treated groups were observed. Histologically, the model group had massive growth of columnar stromal and epithelial cells. CMARE and finasteride attenuated this growth with a resultant thin layer of stromal and epithelial cells similar to the control. PSA levels were significantly lower in the treatment groups. CONCLUSION: CMARE reduces stromal and epithelial cell growth, and subsequently shrinks enlarged prostate. This is the first scientific proof validating the anecdotal evidence of CMARE efficacy in the management of BPH.

Prostate-specific targeting of the aqueous root extract of Croton membranaceus in experimental animals.

Croton membranaceus Müll.Arg. (Euphorbiaceae) is used for benign prostate hyperplasia (BPH) treatment. The study aimed at investigating organs that the aqueous root extracts of C. membranaceus (CMARE) target, which is absent in literature. Twenty-four male Sprague-Dawley rats (100-140 g) were randomly divided into 4 groups. Group 1, the control group received distilled water. Groups 2, 3 and 4 received 30, 150 and 300 mg kg(-1) b.wt CMARE respectively (oral gavage). Rats fed 90 days the standard chow diet ad libitum. Upon sacrifice, major organs were histologically examined and serum prostate-specific antigen (PSA) biochemically determined. Only the prostate was abnormal. Histologically, H&E staining revealed thickness and infoldings of the epithelial cells shrinking with increasing dose. The 30 mg kg(-1) group showed low columnar or flattened epithelium cells, whereas the columnar epithelium infoldings of the 150 mg kg(-1) b.wt and 300 mg kg(-1) b.wt groups were virtually nonexistent. The acini of the control, 30 mg kg(-1) b.wt group and the 150 mg kg(-1) b.wt groups showed clear pinkish secretion. However, secretion of the high-dose group appeared light pink in colour and the stroma cells appeared much darker than all the treated and control group. C. membranaceus targets the prostate with significant PSA reduction (P < 0.01).

Genotoxicity, cytotoxicity and toxicological evaluation of whole plant extracts of the medicinal plant Phyllanthus niruri (Phyllanthaceae).

Phyllanthus niruri is a medicinal plant (commonly known as stone breaker) found in the tropics and other parts of the world. It is known for its capacity to block the formation of calcium oxalate crystals and kidney stone formation in urolithiasis. This plant has been used to treat hyperglycemia, hypertension, pain, and mild cases of malaria. We examined the geno-, cyto- and overall toxicity of P. niruri whole plant ethanolic extract. The extract was administered as a single dose of 30 or 300 mg/kg to laboratory rats by gavage, accompanied by negative (0.9% saline) and positive (10 mg/mL N-ethyl-N-nitrosourea) controls that were injected intramuscularly 48 h after extract administration. The ratio of polychromatic (PCE)/normochromatic erythrocytes (NCE) from femur bone marrow was scored for genotoxicity. Cytotoxicity was determined using descending concentrations (0.2-0.0125 g/mL) of the extract incubated with peripheral blood mononuclear cells. Lactate dehydrogenase release from damaged cells was determined and the CC(50) calculated. Subchronic administration of the extract at 30 or 300 mg/kg was done for 90 days to determine general toxicity. PCE:NCE (%) for the extract and negative control was 63, compared to 168 (positive control). The CC(50) was 26.3 mg/mL and hepato-renal toxicity after subchronic extract administration was nil. We conclude that ethanol extract of P. niruri is not cytotoxic or genotoxic, and is generally non-toxic on subchronic administration.

Acute toxicity studies of Croton membranaceus root extract.

AIM OF THE STUDY: Croton membranaceus root and leaf extracts are used in the Bahamas to aromatize tobacco, in Nigeria to improve digestion, and in Ghana, for benign prostate hyperplasia. Despite claims of success there is paucity of information on its toxicity. The aim of this study was to determine if Croton membranaceus has acute toxicity properties. MATERIALS AND METHODS: Roots were air-dried in a solar dryer for one week before milling. The powder was extracted with 96% ethanol, freeze-dried and re-extracted with distilled water and freeze-dried. 15 male Sprague-Dawley rats (180-200 g) were divided equally into 2 treatment groups [low dose (LD) and high dose (HD)], plus a control group (C). LD and HD received 1500 and 3000 mg/kg b.wt. Croton membranaceus aqueous extract, respectively, one time and observed for 14 days. Haematological [Full Blood Count and haemoglobin (Hb)], biochemical [bilirubin, alanine aminotransferase (ALA), aspartate aminotransferase (AST), total protein, albumin, globulin, alkaline phosphatise (ALP), γ-glutamyltranspetidase (GGT), urea, creatinine, creatinine kinase - Muscle and Brain (CK-MB), creatinine kinase - Total (CK-R)] examinations were performed. RESULTS: Control group's CK-MB (5444±534 U/L) and LD group CK-MB (4014±1016 U/L) were significantly different (p<0.05). Control and the HD group CK-MB (3955±1135 U/L) were significantly different (p<0.05). Both LD and HD CK-R levels (697±197U/L and 732±203 U/L, respectively), were lower than the control (1139±220 U/L) at 48 h and 14 days (p<0.05, p<0.05, respectively). γ-GT levels of the HD group was 4.8±0.4 U/L compared to the Control group value of 0.9±0.2 U/L (p<0.05). CONCLUSIONS: Taking all factors into consideration, Croton membranaceus ingestion does not produce general acute toxicity. However, its creatinine kinase lowering ability could be explored.

Clinical efficacy of a tea-bag formulation of cryptolepis sanguinolenta root in the treatment of acute uncomplicated falciparum malaria.

OBJECTIVE: To evaluate the clinical efficacy and safety of a tea bag formulation of the root of C. sanguinolenta DESIGN: This is a prospective descriptive open trial. SETTING: Patients were recruited from Korle-Bu, Mamprobi and Dansoman Polyclinics. SUBJECTS: Forty-four subjects with uncomplicated malaria were recruited for the study. METHOD: Patients presented with clinical symptoms of malaria. Laboratory investigations conducted included malaria parasite counts, haematological indices and biochemical tests which were obtained before, during and after a 5-day treatment period, up to Day 28 posttreatment. All patients in the study were symptomatic with significant parasitaemia. Patients were given one teabag three times a day, that is, morning, noon and night, for five days of treatment. RESULTS: Fifty percent of the patients were cleared of their P. falciparum parasitaemia by 72 hours, and all by Day 7. Presenting symptoms of fever, chills, nausea and vomiting cleared rapidly, all by Day 3, but resolution of haematological and biochemical abnormalities associated with malaria was generally slow, a feature seen in malaria post-treatment. The overall cure rate was 93.5% due to two cases of recrudescence on Days 21 and 28. The laboratory findings did not suggest any toxicity. CONCLUSION: On the basis of fever clearance and disappearance of parasitaemia by Day 7, the formulation has been shown to be non-toxic and highly effective in the treatment of acute uncomplicated malaria.

Pharmacokinetics of enrofloxacin given by the oral, intravenous and intramuscular routes in broiler chickens.

Enrofloxacin was given to broiler chickens, 3 groups of 6 birds each, at a dose of 5 mg/kg. Routes of administration were intravenous (i.v.), intramuscular (i.m.) and oral (p.o.) and blood samples were collected from the jugular vein for determination of serum drug levels over a 54-hour period after administration. Drug levels were determined using Bacillus subtilis spore suspension on Meuller-Hinton antibiotic medium. Intravenous administration produced drug levels which followed a bi-exponential decay according to the model C = 101e(-1.84(t)) + 1.30e(-0.06(t)). After i.m. administration, the mean Cmax observed (2.01 microg/mL) occurred at 1 h and levels were detected for up to 48 h. The mean time to maximum concentration (Tmax) for the birds occurred at 0.79 h. The model describing serum concentrations after i.m. administration was C = 1.35e(-0.48(t)) + 1.27e(-0.07(t)) - 2.06e(-2.1(t)). Serum concentrations after oral administration were lower and the mean +/- standard error of mean, of the maximum concentrations (Cmax) was 0.99 microg/mL at 2 h after administration. The mean residence times after the 3 routes of administration were not significantly different and ranged from 12.5-13.7 h. Bioavailability by the oral route was 80.1%. Dialysis of chicken plasma vs saline indicated that the protein binding was 22.7%.