Investigating the phytoavailability of metals in roots of Croton macrostachyus and Phytolacca dodecandra: induced rhizosphere processes.

Medicinal plants are key component of complementary and alternative medicine, and constitute the first therapeutic strategy for majority of the global population, particularly in developing nations. Croton macrostachyus and Phytolacca dodecandra are among such plants, widely used to treat various diseases. The present study aimed at investigating the elemental composition of both plant root samples collected from four districts in the Central Gondar Zone, Northwest Ethiopia. An optimized wet-digestion procedure was applied with 0.5 g of root sample using HNO3: HCl: H2O2 (v/v) in the ratio of 8:2:1 at 240 °C for 3:00 h (for P. dodecandra), and 5:2:2 at 300 °C for 3:00 h (for C. macrostachyus). Then, concentrations of metals in the digested samples were determined using a flame atomic absorption spectrophotometer; and the validity of the optimized procedure was evaluated by spiking experiments. Results showed good accuracy and repeatability of the methods with percent of recoveries ranging from 85 to 103%. The mean concentrations of metals in C. macrostachyus root samples were higher than their P. dodecandra counterparts, and the distribution of metals showed the following decreasing order: Mg > Ca ≈ Fe > Mn > Zn > Cu > Cr. One-way ANOVA revealed no significant differences (p > 0.05) between the mean concentrations of the same metals in both plant root samples, except for Mg and Cr. Correlation matrix, principal component and cluster analyses suggested that accumulation of metals in the root samples originated from both natural and anthropogenic sources. In conclusion, the roots of both medicinal plants contained substantial amounts of metals, thereby serving as valuable sources of essential elements.

In Vitro Evaluation of the Effects of Selected Plants on the Growth of the Mycelial Form of Histoplasma capsulatum Variety farciminosum in Ethiopia.

Epizootic lymphangitis is prevalent in equines in Ethiopia, causing remarkable economic and welfare impacts but often neglected. Lack of effective treatment contributed to its continued occurrence, and hence, search for an effective treatment should be considered a priority area to minimize its impacts. Previous ethnobotanical studies have reported that Curcuma longa, Phytolacca dodecandra, and Datura stramonium were used to treat cutaneous fungal infections and reduce their incidence. The treatment effects of these plants against epizootic lymphangitis should be studied. The in vitro growth inhibitory effects of methanol extracts of the root of C. longa, berry of P. dodecandra, and leaf of D. stramonium were evaluated. Histoplasma capsulatum var farciminosum was isolated from clinical cases of epizootic lymphangitis in carthorses in central Ethiopia. The nested polymerase chain reaction was used to confirm the identity of the isolates. Serial twofold dilutions of the extract of berries of P. dodecandra and leaves of D. stramonium were done in sterile water, whereas dilution of the extract of roots of C. longa was done in dimethylsulphoxide. The effects of the plants on the growth of Histoplasma capsulatum var farciminosum were assessed by agar dilution assay. Culture media with no antifungal agent and media containing ketoconazole served as negative and positive control, respectively. The methanol extract of C. longa showed inhibitory effects at concentrations ranging from 0.07 to 5 mg/mL. Similarly, the methanol extract of P. dodecandra showed growth inhibitory effects at concentrations ranging from 0.156 to 5 mg/mL. That is, the growth inhibitory concentration of C. longa was 0.07 mg/mL, whereas that of P. dodecandra was 0.156 mg/mL. In contrast, D. stramonium showed no inhibitory effect. This preliminary observation showed that methanol extracts of C. longa and P. dodecandra showed inhibitory effects on the growth of Histoplasma capsulatum var farciminosum requiring further repeated in vitro evaluation so as to generate adequate evidence, which would justify in vivo trials.

Larvicidal effect of Endod (Phytolacca dodecandra) seed products against Anopheles arabiensis (Diptera: Culicidae) in Ethiopia.

OBJECTIVE: The purpose of the present study was to determine the larvicidal effect of 'Endod' (Phytolacca dodecandra) seed products on Anopheles arabiensis, in Ethiopia. RESULTS: Experimental study was conducted using a total of 2400 third instars larvae of A. arabiensis. The seed products P. dodecandra showed larvicidal activity against 3rd-stage larvae of both the laboratory and field population of A. arabiensis. The LC99 values for P. dodecandra's seed powder and its extract form against the laboratory reared larvae were 121.07, and 616.46 mg/l, respectively. The LC50 and LC95 values were also determined.

Homeopathic therapy for sleep bruxism in a child: Findings of a 2-year case report.

Bruxism is a sleep disorder characterized by grinding and biting teeth with multifactorial etiology, resulting in deleterious effects on teeth, periodontium, and temporomandibular joint. There is a lack of scientific evidence evaluating the effectiveness of medicines in treating this parafunction. The present case report was drafted under the rules of CARE checklist. An 8-year-old male patient with sleep bruxism and associated symptoms received a combined homeopathic therapy of Phytolacca decandra 12c and Melissa officinalis 12c for 2 months. After this period of combined homeopathic therapy, the bruxism and associated symptoms completely disappeared. After 2 years of clinical follow-ups, the patient had no recurrences. The use of homeopathic therapy was successful and should be seen as an alternative to treat sleep bruxism and its associated symptoms in children.

Evaluation of Endod (Phytolacca dodecandra: Phytolaccaceae) as a Larvicide Against Anopheles arabiensis, the Principal Vector of Malaria in Ethiopia.

Malaria control methods rely mostly on adult mosquito control using insecticide-treated nets and indoor residual spraying with insecticides. Plants such as endod (Phytolacca dodecandra) can potentially be used for the control of mosquito larvae as a supplement to adult control methods. Following the discovery of endod, a molluscicide plant, more than 5 decades ago in Ethiopia, subsequent studies have shown that its potency can further be increased by simple procedures such as aging endod berry powder in water. This study was conducted to evaluate the killing effect of fresh and aged endod solution against 4th-stage larvae of Anopheles arabiensis. Laboratory-reared An. arabiensis larvae exposed to different concentrations of endod preparation using distilled or spring water had 50% lethal concentration (LC(50))  =  49.6 ppm and 90% lethal concentration (LC(90))  =  234 ppm for fresh and LC(50)  =  36.4 ppm and LC(90)  =  115.7 ppm for the aged endod solution in distilled water against the laboratory population. Against field-collected larvae of the same species, aged preparations in habitat water resulted in higher LC(50) (472.7 ppm) and LC(90) (691 ppm) values, with only a slight improvement over fresh preparations in habitat water (LC(50)  =  456.2 ppm; LC(90)  =  896.1 ppm). In general, although aged preparations of endod required lower concentrations than fresh to kill at least 90% of the larvae, these concentrations were much higher (12-70×) than that required for schistosome-transmitting snails.

Anti-proliferative effects of homeopathic medicines on human kidney, colon and breast cancer cells.

OBJECTIVE: Homeopathy is controversial, due to the claims made for very high dilutions. Although several theories are proposed to understand the mechanisms of action, none are scientifically verified. This study aimed to investigate the efficacy of the selected homeopathic medicines in specific in vitro cancer models. METHODS: We assessed the cytotoxic activity of selected homeopathic medicines in mother tincture (MT), and ultramolecular dilution (30C, 200C, 1M and 10M) against cell lines deriving from tumors of particular organs, Sarsaparilla (Sars) on ACHN cells (human renal adenocarcinoma), Ruta graveolens (Ruta) on COLO-205 (human colorectal carcinoma), and Phytolacca decandra (Phyto) on MCF-7 (human breast carcinoma). Sars was also tested against Madin-Darby canine kidney (MDCK) cells (a non-malignant cell line). Cytotoxicity was measured using the 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) method, anti-proliferative activity by trypan blue exclusion assay, apoptosis determined by dual staining the cells with ethidium bromide (EB) and acridine orange (AO) dyes. RESULTS: MTs and ultra-diluted preparations of the three homeopathic medicines had highly significant effects in the respective cancer cell lines, producing cytotoxicity and a decrease in cell proliferation. The effects were greatest with the MTs, but in all cases and persisted, although to a lesser degree in the ultra-diluted molecular preparations. Sars showed no effect on MDCK cells. In the homeopathic medicine treated cultures, hallmarks of apoptosis were evident including, cell shrinkage, chromatin condensation and DNA fragmentation. CONCLUSION: This study provides preliminary laboratory evidence indicating the ability of homeopathic medicines as anticancer agents. Further studies of the action of these homeopathic remedies are warranted.

Biosynthesized silver nanoparticles by ethanolic extracts of Phytolacca decandra, Gelsemium sempervirens, Hydrastis canadensis and Thuja occidentalis induce differential cytotoxicity through G2/M arrest in A375 cells.

The capability of crude ethanolic extracts of certain medicinal plants like Phytolacca decandra, Gelsemium sempervirens, Hydrastis canadensis and Thuja occidentalis used as homeopathic mother tinctures in precipitating silver nanoparticles from aqueous solution of silver nitrate has been explored. Nanoparticles thus precipitated were characterized by spectroscopic, dynamic light scattering, X-ray diffraction, atomic force and transmission electron microscopic analyses. The drug-DNA interactions of silver nanoparticles were analyzed from data of circular dichroism spectroscopy and melting temperature profiles using calf thymus DNA (CT-DNA) as target. Biological activities of silver nanoparticles of different origin were then tested to evaluate their effective anti-proliferative and anti-bacterial properties, if any, by exposing them to A375 skin melanoma cells and to Escherichia coli C, respectively. Silver nanoparticles showed differences in their level of anti-cancer and anti-bacterial potentials. The nanoparticles of different origin interacted differently with CT-DNA, showing differences in their binding capacities. Particle size differences of the nanoparticles could be attributed for causing differences in their cellular entry and biological action. The ethanolic extracts of these plants had not been tested earlier for their possible efficacies in synthesizing nanoparticles from silver nitrate solution that had beneficial biological action, opening up a possibility of having therapeutic values in the management of diseases including cancer.

Evaluation of berries of Phytolacca dodecandra for growth inhibition of Histoplasma capsulatum var. farciminosum and treatment of cases of epizootic lymphangitis in Ethiopia.

OBJECTIVE: To evaluate the berries of Phytolacca dodecandra (P. dodecandra) for its effect on Histoplasma capsulatum var. farciminosum (HCF) and for the treatment of cases of epizootic lymphangitis (EL). METHODS: Samples were collected from un-ruptured nodules of cases of EL at Debre Zeit and Akaki (central Ethiopia). Mycological culture and isolation of HCF were performed at the Aklilu Lemma Institute of Pathobiology. Phytochemical screening was done for n-butanol extract of P. dodecandra to detect alkaloids, saponins, phenolic compounds and flavonoids. The minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of aqueous and n-butanol extracts of P. dodecandra against HCF were determined by agar dilution assay. For the in vivo trial, 5% simple ointment was prepared from n-butanol extract and applied topically to 24 (twelve early and twelve moderate) cases of EL. RESULTS: Phytochemical screening showed that n-butanol extract of P. dodecandra was positive for alkaloids, saponins and phenolic compounds but negative for flavonoids. The MICs of n-butanol and aqueous extracts of P. dodecandra were (0.039%-0.078%) and (0.625%-1.250%), respectively. The MFCs of n-butanol and aqueous extracts of P. dodecandra were (0.078%-0.156%) and (1.250%-2.500%), respectively. The MIC and MFC of ketoconazole (positive control) was (1.200×10(-5)%-2.500×10(-5)%) and (5.000×10(-5)%-1.000×10(-4)%), respectively while growth was observed on free medium (negative control). From the total of 24 treated cases of EL, 14 (58.3%) responded to treatment; however, 10 (41.7%) did not respond to treatment. There was no significant difference in the degree of response to treatment between early and moderate cases (χ(2)=0.686; P=0.408). CONCLUSIONS: It can be concluded that n-butanol extract of P. dodecandra demonstrates antifungal effects while the aqueous extract shows no antifungal activity.

Equine histoplasmosis: treatment trial in cart horses in Central Ethiopia.

Therapeutic effects of Sodium Iodide (NaI), Potassium Iodide (KI), ground berries of "Endod" (Phytolacca dodecandra) and Penstrip were evaluated on 70 cases of equine hitoplasmosis (EH). Response to each treatment was assessed using clinical examination of the lesions. Statistically significant difference (P = 0.0036) in therapeutic effect was observed among the different remedies. Cases treated either with a combination of NaI and Penstrip (F = 6.34, P = 0.004) or "Endod" and Penstrip (F = 3.64, P = 0.031) demonstrated significant response. The difference in response to treatment between early and advanced cases of EH was statistically significant (t = 2.22, P = 0.0148).

Laboratory evaluation of Ethiopian local plant Phytolacca dodecandra extract for its toxicity effectiveness against aquatic macroinvertebrates.

In this study, we evaluated the toxicity effectiveness of berries crude extract of Endod [vernacular name (local native language, Amharic); Phytolacca dodecandra] against aquatic macroinvertebrates Baetidae (Mayflies) and Hydropsychidae (Caddisflies), under laboratory conditions. In Ethiopia, toxic plant, berries of Phytolacca dodecandra are being commonly used for washing clothes and to control fresh water snails. Macroinvertebrates are useful biological indicators of change in the aquatic ecosystems. The present study clearly revealed that the LC50 and LC90 values for berries crude extract of Phytolacca dodecandra against Baetidae were 181.94 and 525.78 mg/l and lethal doses (LC50 and LC90) required for Hydropsychidae were 1060.69 and 4120.4 mg/l respectively. The present investigation demonstrated that Baetidae was more susceptible than Hydropsychidae, even at shorter exposure period of 2 h. From our preliminary investigation the toxicity effectiveness of crude extracts of Phytolacca dodecandra has been clearly shown. In addition, it requires further explorations which address both the toxicity activity and the active principles that are responsible for its toxicity effectiveness. Ultimately, the release/introduction of Phytolacca dodecandra plant berries extracts into the river/streams leads to disruption of food chain in the aquatic ecosystem. Therefore, at this moment preserving the aquatic ecosystem is extremely essential and inevitable.

In vitro activity of Phytolacca dodecandra (Endod) against dermatophytes.

There is a need for better and less costly treatment of superficial mycotic infections, and one approach might be testing of'traditionally used plants for their antifungal activities as potentials for drug development. The aim of the present study was to determine the in vitro activity of Phytolacca dodecandra (Endod) on various species of dermatophytes and yeasts of medical importance. The aqueous extract from the plant P. dodecandra was tested for its antifungal activity against 33 dermatophyte and yeast strains. The minimum inhibitory concentration (MIC) was determined by adopting the broth microdilution method for testing of conidium forming filamentous fungi according to the NCCLS M38-P proposed standard with some modifications. The MIC of P. dodecandra against the dermatophytes tested ranged from 19.5 mg/l to 156.0 mg/l, while for all the yeasts the MIC was >500 mg/l. The minimum inhibitory concentration for 50% (MIC50) of the dermatophyte strains was 62.5 mg/l. The extract showed fungicidal activity against the dermatophytes that ranged from 19.5 to 312.5 mg/l. No activity was observed against the yeasts. From our preliminary results antifungal activity of P. dodecandra has been shown. This should he strengthened with more extensive studies which address both the antifungal activity and the active principle that is responsible for its fungicidal activity.

Control of Biomphalaria pfeifferi population and schistosomiasis transmission in Ethiopia using the soap berry endod (Phytolacca dodecandra), with special emphasis on application methods.

The endod (Phytolacca dodecandra)-based schistosomiasis mansoni control project was implemented in Ethiopia between 1994 and 1999. The aim was to develop an effective, cheap and sustainable method of controlling schistosomiasis. First, different formulations of the Ethiopian endod strain 44 (E-44) were compared for potency in the laboratory. Secondly, spray and drip-feeding methods were compared for simplicity and effectiveness in the field. Lastly, the efficacy of endod powder soap was compared with the endod spray method. In Bati stream, endod powder soap was distributed to the residents every weekend at laundry sites. In Worke stream, endod was sprayed along a 1-km stretch of the stream at 3-month intervals. No endod was applied in Harbu stream. The immediate and long-term effects of endod application on the snail population and schistosomal infection were determined. Using the spray method, 100% snail mortality could be obtained, and it was simpler and more effective than the drip-feeding method. Snail mortality ranged from 20 to 100% using endod soap. There was a progressive decline in the snail population and infection in Bati stream compared with Worke stream, mainly due to sustained use of endod soap. The advantages and disadvantages of the different endod delivery systems are discussed.

Control of Schistosoma mansoni by the soapberry Endod (Phytolacca dodecandra) in Wollo, northeastern Ethiopia: post-intervention prevalence.

OBJECTIVES: To record the effect of Endod soap and spraying of soaked Endod suspension on the prevalence of human schistosomiasis. DESIGN: A cross-sectional epidemiological study in which pre- and post-intervention parasitological results were compared. SETTING: Kemise, Bati and Harbu towns in northeastern Ethiopia. SUBJECTS: The study subjects included all members of the five per cent households systematically selected from the three towns. RESULTS: In Kemise town, where suspension of ground Endod was sprayed on the stream containing infected snails, the prevalence of the disease was reduced from 59% to 53% and the mean intensity of infection was reduced from 239 eggs per gram (EPG) of faeces to 99 EPG (p < 0.05). In Bati town where Endod soap approach was used, the respective reduction in the prevalence and intensity of infection was from 51% to 43% and from 195 EPG to 162 EPG (p < 0.05). There was also a significant reduction of the disease in the control town probably due to the effects of praziquantel treatment and other factors. CONCLUSION: The reduction achieved in the prevalence and intensity of schistosomiasis after an intervention period of four years was limited. This observation corroborates the fact that molluscicides must always be considered as supplementary to chemotherapy in the control of schistosomiasis. Although both approaches can be used, the spraying approach appears to be simpler and more feasible because two or three times yearly application of Endod suspension would suppress snail population and reduce transmission. Nevertheless, the choice as to what approach to use must be made on the basis of community preference, and for some soap-effect of Endod would be attractive

Endod: safety evaluation of a plant molluscicide.

Toxicity tests, in accordance with the Minimal Data Requirements (Tier 1) of the OECD Guidelines for Pre-Market Chemicals, were conducted on a standard extract (Endod-S) from the unripe berries of Phytolacca dodecandra, a potent botanical molluscicide of potential importance in the control of schistosomiasis. In acute mammalian toxicity tests, except for the eye irritation toxicity test which indicated severe irritancy, all test results were classified as either nontoxic or slightly toxic. Eye protection is therefore recommended during berry crushing and handling of dry powders. Ecotoxicity tests indicated that Endod is no more toxic than currently recommended synthetic molluscicides; however, environmental fate and additional local ecotoxicity tests are recommended for nontarget aquatic organisms present in the endemic situations of field trials. Given these toxicological data and recognizing the need for an affordable, locally cultivated, botanical molluscicide, it is concluded that field trials of Endod in schistosomiasis control are now justifiable.

Evaluation of the mutagenic potential of endod (Phytolacca dodecandra), a molluscicide of potential value for the control of schistosomiasis.

Extracts of the fruit of Phytolacca dodecandra (endod) demonstrate molluscicidal and other biological activities. Since this plant is indigenous to some countries where schistosomiasis is a common problem, it has been proposed that it may be socioeconomically feasible to employ endod as an aid in the control of this disease through its use to control the snail vector. As an initial step in the safety assessment of this substance, its mutagenic potential was determined utilizing Salmonella typhimurium strain TM677. The seeds and fruit of Phytolacca americana, also molluscicidal, were additionally evaluated for mutagenic potential. Using a variety of conditions, no mutagenic activity could be demonstrated for any of the extracts tested. Thus, subject to the results of future safety assessment, endod remains a viable candidate as a useful molluscicide.

Control of schistosomiasis in Adwa, Ethiopia, using the plant molluscicide endod (Phytolacca dodecandra).

Control of Schistosoma mansoni transmission in a town in northern Ethiopia was attempted using the natural product, endod (Phytolacca dodecandra), and its use compared with niclosamide. Within a five year period prevalence was reduced from 61.5 to 36.4% in the area where endod was used. Most of this decline was accounted for by the reduction in prevalence among 1-6 year old subjects. The practical use of endod as a molluscicide was demonstrated to have a performance which compared favourably with the commercial product, and although more expensive than niclosamide still at minimal annual cost (US + 0.06 per capita).