Assessment of drug delivery and anticancer potentials of nanoparticles-loaded siRNA targeting STAT3 in lung cancer, in vitro and in vivo.

Activation of signal transducer and activator of transcription3 (STAT3) is a hallmark of several types of cancer. Failure to inhibit STAT3 expression by injection of siRNA for STAT3 directly to Balb/c mice led us to adopt alternative means. We formulated nanoparticle-based encapsulation of siRNA (NsiRNA) with polyethylenimine (PEI) and poly(lactide-co-glycolide) (PLGA) and characterized them. The siRNA treated and NsiRNA-treated cells were subjected separately to different assay systems. We also checked if NsiRNA could cross the blood brain barrier (BBB). Cell viability reduced dramatically in A549 cells after NsiRNA administration (23.89% at 24 h), thereby implicating considerable silencing of STAT3 by NsiRNA, but not after siRNA administration. Compared to controls, a significant decrease in expression of IL-6 and the angiogenic factor (VEGF) and increase in Caspase 3 activity was observed with corresponding regression in tumor growth in mice treated with NsiRNA. NsiRNA induced apoptosis of cells and arrested cells at G1/G0 stage, both in vitro and in vivo. Apoptosis was also verified by Annexin-V-FITC/Propidium-iodide staining. NsiRNA could cross blood brain barrier. Overall results revealed PEI-PLGA to be a promising carrier for delivery of siRNA targeting STAT3 expression, which can be utilized as an effective strategy for cancer therapy.

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.

Green synthesis, characterization and anticancer potential of platinum nanoparticles Bioplatin.

OBJECTIVE: In the present study, the anticancer potential of platinum nanoparticles Bioplatin is explored and the mode of interactions of Bioplatin with calf thymus DNA and honey was analyzed. METHODS: Bioplatin was synthesized with the help of green nanotechnology and characterized by particle size, zeta potential and surface morphology. The interaction of Bioplatin with DNA and honey was also checked with the help of circular dichroism spectroscopy and Fourier-transform infrared spectroscopy, respectively. The anticancer potential of Bioplatin was evaluated on peripheral blood mononuclear cells and A375 cells in vitro by analyzing results of MTT (3-(4,5)-dimethyl-thiahiazo-(-z-y1)-3,5-di-phenytetrazoliumromide), fluorescence microscopic studies and DNA fragmentation assay. RESULTS: Bioplatin exhibited a small particle size of 137.5 nm and a surface charge of -35.8 mV. Bioplatin interacted with DNA and brought in effective changes in structure and conformation of DNA, and formed a new complex that increased its stability of DNA intercalated with the base pair of DNA. In vitro studies demonstrated that Bioplatin arrested cell proliferation, and induced chromatin condensation and internucleosomal DNA fragmentation. CONCLUSION: Bioplatin induces apoptosis in cancer cells and may have some beneficial effect against human carcinoma. It interacts with DNA, brings stabilization to DNA, and thus prevents the replication of DNA.

Rapid green synthesis of silver nanoparticles from silver nitrate by a homeopathic mother tincture Phytolacca Decandra.

OBJECTIVE: To examine if a homeopathic mother tincture (Phytolacca Decandra) is capable of precipitating silver nanoparticles from silver nitrate (AgNO(3)) and to characterize the biosynthesized nanoparticles for evaluating their biological activities. METHODS: A total of 100 mg of AgNO(3) was added to 20mL of Milli-Q water and stirred vigorously. Then 5mL of the homeopathic mother tincture of Phytolacca Decandra (ethanolic root extract of Phytolacca decandra) was added and stirred continuously. Reduction took place rapidly at 300K and completed in 10 min as shown by stable light greenish-yellow color of the solution which gave colloid of silver nanoparticles. The colloid solution was then centrifuged at 5000×g to separate the nanoparticles for further use. The nanoparticles were characterized by spectroscopic analysis, particle size analysis and zeta potential measurements, and morphology was analyzed by atomic force microscopy. The drug-DNA interaction was determined by circular dichroism spectrophotometry and melting temperature profiles by using calf thymus DNA as the target. The biological activities were determined using a cancer cell line A549 in vitro and using bacteria Escherichia coli and fungus Saccharomyces cerevisiae as test models. RESULTS: Phytolacca Decandra precipitated silver nanoparticles in ambient conditions. The nanoparticles had 91 nm particle size, with polydispersity index of 0.119 and zeta potential of -15.6 mV. The silver nanoparticles showed anticancer and antibacterial properties, but no clear antifungal properties. CONCLUSION: This could be a novel environment-friendly method to biosynthesize silver nanoparticles using a cost-effective, nontoxic manner. The homeopathic mother tincture may utilize this property of nano-precipitation in curing diseases or disease symptoms.

Thujone-Rich Fraction of Thuja occidentalis Demonstrates Major Anti-Cancer Potentials: Evidences from In Vitro Studies on A375 Cells.

CRUDE ETHANOLIC EXTRACT OF THUJA OCCIDENTALIS (FAM: Cupressaceae) is used as homeopathic mother tincture (TOΦ) to treat various ailments, particularly moles and tumors, and also used in various other systems of traditional medicine. Anti-proliferative and apoptosis-inducing properties of TOΦ and the thujone-rich fraction (TRF) separated from it have been evaluated for their possible anti-cancer potentials in the malignant melanoma cell line A375. On initial trial by S-diphenyltetrazolium bromide assay, both TOΦ and TRF showed maximum cytotoxic effect on A375 cell line while the other three principal fractions separated by chromatography had negligible or no such effect, because of which only TRF was further characterized and subjected to certain other assays for determining its precise anti-proliferative and apoptotic potentials. TRF was reported to have a molecular formula of C(10)H(16)O with a molecular weight of 152. Exposure of TRF of Thuja occidentalis to A375 cells in vitro showed more cytotoxic, anti-proliferative and apoptotic effects as compared with TOΦ, but had minimal growth inhibitory responses when exposed to normal cells (peripheral blood mononuclear cell). Furthermore, both TOΦ and TRF also caused a significant decrease in cell viability, induced inter-nucleosomal DNA fragmentation, mitochondrial transmembrane potential collapse, increase in ROS generation, and release of cytochrome c and caspase-3 activation, all of which are closely related to the induction of apoptosis in A375 cells. Thus, TRF showed and matched all the anti-cancer responses of TOΦ and could be the main bio-active fraction. The use of TOΦ in traditional medicines against tumors has, therefore, a scientific basis.

Poly (lactide-co-glycolide) acid nanoencapsulation of a synthetic coumarin: cytotoxicity and bio-distribution in mice, in cancer cell line and interaction with calf thymus DNA as target.

Several naturally occurring coumarin compounds, including scopoletin (7 hydroxy-6 methoxycoumarin), of plant origin have been reported to have anti-cancer potentials. A related but chemically synthesized coumarin, 4-methyl-7-hydroxy coumarin (SC), was also shown to have similar anti-cancer potentials. In the present study, to test if nano-encapsulated SC could be a more potent anti-cancer agent, we encapsulated SC with poly lactide-co-glycolide acid (PLGA) nanoparticles (Nano Coumarin; NC) and tested its potentials with a variety of protocols. NC demonstrated greater efficiency of drug uptake and showed anti-cancer potentials in melanoma cell line A375, as revealed from scanning electronic and atomic force microscopies. To test its possible interaction with target DNA, the combined data of circular dichroism spectra (CD) and melting temperature profile (T(m)) of calf thymus DNA treated with NC were analyzed. Results indicated a concentration dependent interaction of NC with calf thymus DNA, bringing in effective change in structure and conformation, and forming a new complex that increased its stability. Particle size and morphology of NC determined through polydispersity index and zeta potential using dynamic light scattering qualified NC to be a more potent anti-cancer agent than SC. Further, SC and NC showed negligible cytotoxic effects on normal skin cells and peripheral blood mononuclear cells of mice. Distribution assay of PLGA nanoparticles in different tissues like brain, heart, kidneys, liver, lungs, and spleen in mice revealed the presence of nanoparticles in different tissues including brain, indicating that the particles could cross the blood brain barrier, significant information for drug design.

Anticancer potentials of root extract of Polygala senega against benzo[a]pyrene-induced lung cancer in mice.

OBJECTIVE: To evaluate anticancer potentials of Polygala senega on lung cancer induced by benzo[a]pyrene (B[a]P) in mice. METHODS: Swiss albino mice were divided into five groups with each containing six animals. Group 1 served as control, and the animals received olive oil as vehicle. Group 2 animals were treated with B[a]P (50 mg/kg body weight dissolved in olive oil) orally twice a week for four consecutive weeks. Group 3 animals were fed B[a]P as in group 2 and 48% alcohol (since the vehicle of the remedy was alcohol). Group 4 animals were B[a]P-intoxicated mice (as in group 2) which were additionally fed ethanolic extract of Polygala senega (EEPS) daily for 16 weeks. EEPS treatment started after the first dose of B[a]P. Group 5 animals were treated with EEPS alone for 16 weeks to test cytotoxicity of EEPS if any. Mice were sacrificed after 16 weeks and the following parameters were assessed: the anti-oxidant activity measured by 2,2-diphenyl-1-picrylhydrazyl free radical assay, tumor incidence, lung weight and body weight, DNA damage evaluation by comet assay and enzyme-linked immunosorbent assay (ELISA); toxicity biomarkers like catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, lipid peroxidation (LPO) and total thiol content were also detected. RESULTS: Treatment with EEPS increased the final body weight and significantly decreased the lung weight in group 4 mice (P<0.01) compared with group 3 mice. Comet assay showed that EEPS-treated mice in group 4 presented a decrease of DNA damage significantly (P<0.01) in lung tissues. There was a significant increase observed in the level of p53 in group 4 as compared with group 3 (P<0.01) detected by ELISA. A highly significant increase in tissue LPO with concomitant decrease in the activity of anti-oxidants was observed in group 2 and group 3 mice (P<0.05) compared with the control mice. These adverse changes were reversed significantly in group 4 mice (P<0.01). CONCLUSION: Chemopreventive potentials of Polygala senega against chemically induced lung cancer in mice are confirmed.

Anticancer Potentials of Root Extract of Polygala senega and Its PLGA Nanoparticles-Encapsulated Form.

Ethanolic extract of Polygala senega (EEPS) had little or no cytotoxic effects on normal lung cells, but caused cell death and apoptosis to lung cancer cell line A549. In the present paper, ethanolic root extract of P. senega (EEPS) was nanoencapsulated (size: 147.7 nm) by deploying a biodegradable poly-(lactic-co-glycolic) acid (PLGA). The small size of the NEEPS resulted in an enhanced cellular entry and greater bioavailability. The growth of cancer cells was inhibited better by NEEPS than EEPS. Both EEPS and NEEPS induced apoptosis of A549 cells, which was associated with decreased expression of survivin, PCNA mRNA, and increased expression of caspase-3, p53 mRNAs of A549 cells. The results show that the anticancer potential of the formulation of EEPS-loaded PLGA nanoparticles was more effective than EEPS per se, probably due to more aqueous dispersion after nanoencapsulation. Therefore, nanoencapsulated ethanolic root extract of P. senega may serve as a potential chemopreventive agent against lung cancer.

Modulation of Signal Proteins: A Plausible Mechanism to Explain How a Potentized Drug Secale Cor 30C Diluted beyond Avogadro's Limit Combats Skin Papilloma in Mice.

In homeopathy, ability of ultra-high diluted drugs at or above potency 12C (diluted beyond Avogadro's limit) in ameliorating/curing various diseases is often questioned, particularly because the mechanism of action is not precisely known. We tested the hypothesis if suitable modulations of signal proteins could be one of the possible pathways of action of a highly diluted homeopathic drug, Secale cornutum 30C (diluted 10(60) times; Sec cor 30). It could successfully combat DMBA + croton oil-induced skin papilloma in mice as evidenced by histological, cytogenetical, immunofluorescence, ELISA and immunoblot findings. Critical analysis of several signal proteins like AhR, PCNA, Akt, Bcl-2, Bcl-xL, NF-κB and IL-6 and of pro-apoptotic proteins like cytochrome c, Bax, Bad, Apaf, caspase-3 and -9 revealed that Sec cor 30 suitably modulated their expression levels along with amelioration of skin papilloma. FACS data also suggested an increase of cell population at S and G2 phases and decrease in sub-G1 and G1 phages in carcinogen-treated drug-unfed mice, but these were found to be near normal in the Sec cor 30-fed mice. There was reduction in genotoxic and DNA damages in bone marrow cells of Sec Cor 30-fed mice, as revealed from cytogenetic and Comet assays. Changes in histological features of skin papilloma were noted. Immunofluorescence studies of AhR and PCNA also suggested reduced expression of these proteins in Sec cor 30-fed mice, thereby showing its anti-cancer potentials against skin papilloma. Furthermore, this study also supports the hypothesis that potentized homeopathic drugs act at gene regulatory level.

In vitro and in vivo studies demonstrate anticancer property of root extract of Polygala senega.

Polygala senega is extensively used in traditional systems of medicine against various lung diseases including cancer. In the present study we tested the anticancer potentials of ethanolic extract of roots of P. senega (generally used as a homeopathic drug) in a mammalian model, where mice, in vivo, were treated chronically with benzo[a] pyrene and in vitro where lung adenocarcinoma cell line (A549) were used. We deployed various parameters like cell viability assay, chromatin condensation studies with Hoechst 333258 staining, and maintained suitable controls. To understand the possible signal transduction pathways, expression of various signal proteins such as Aryl Hydrocarbon receptor (AhR), cytochrome P450 (CYP1A1), Bcl-2, proliferating cell nuclear antigen (PCNA), Bax and Caspase-3 was studied. Additionally, reverse transcriptase polymerase chain reaction analysis of AhR, p53, PCNA and ß-actin (housekeeping) genes was made. Immunohistochemical localization of PCNA proteins was also conducted in vivo. Feeding of root extract of P. senega to mice (at the rate of 50 mg/kg and 100 mg/kg bw) chronically treated with the carcinogen (50 mg/kg bw dissolved in olive oil) showed positive modulation in expression of signal proteins. Upregulation of apoptotic signals such as p53, Caspase-3 and Bax, and downregulation of AhR, cytochrome P450 (CYP1A1), Bcl-2 and PCNA were observed. Addition of root extract of Polygala Senega (at doses of 50 µg and 100 µg) into culture medium containing A549 cells induced recovery of decreased cell viability and increased chromatin fragmentation (apoptosis). Therefore, results of both in vivo and in vitro studies scientifically validate its potential use as an anticancer agent, particularly against lung cancer, and provide important information potentially helpful in drug designing.

Polymeric nanoparticle encapsulation of a naturally occurring plant scopoletin and its effects on human melanoma cell A375.

OBJECTIVE: We formulated nano-encapsulation of a naturally occurring coumarin-scopoletin (7-hydroxy-6-methoxy coumarin, HMC, C(10)H(8)O(4)), isolated from plant Gelsemium sempervirens having anticancer potentials, with a bio-adhesive agent -polylactic-co-glycolic acid (PLGA) and tested if its cellular uptake, bioavailability and apoptotic (anticancer) potentials could thus be increased vis-a-vis unencapsulated HMC. METHODS: A375 melanoma cancer cells were used for testing cellular entry and anticancer potentials of HMC and nano-7-hydroxy-6-methoxy coumarin (NHMC) through several standard protocols. Characterization of NHMC was done by dynamic light scattering for determination of particle size, polydispersity index (PDI), and zeta potential. Surface morphology of nanoparticles was determined by scanning electron microscopy and atomic force microscopy. RESULTS: HMC was encapsulated with more than 85% entrapment efficiency, the average particle size of NHMC being less than 110 nm and a PDI 0.237, which resulted in enhanced cellular entry and greater bioavailability. NHMC showed a faster cellular uptake (15 min) than its unencapsulated counterpart (30 min). Study of signal molecules through mRNA expressions revealed that NHMC caused down-regulation of cyclin-D1, proliferating cell nuclear antigen (PCNA), survivin and Stat-3, and up-regulation of p53 and caspase-3, that in turn induced a greater number of apoptosis vis-a-vis unencapsulated HMC. CONCLUSION: The formulation yielded small-sized NHMC by biodegradable PLGA that took less time for cellular entry, and caused more apoptosis to cancer cells, but apparently had negligible cytotoxicity against normal skin cells. Nano-encapsulation of bioactive plant ingredients can be a strategy worth trying for designing effective chemopreventive drug products.

Anti-oncogenic potentials of a plant coumarin (7-hydroxy-6-methoxy coumarin) against 7,12-dimethylbenz [a] anthracene-induced skin papilloma in mice: the possible role of several key signal proteins.

OBJECTIVE: Anti-cancer potentials of scopoletin (7-hydroxy-6-methoxy coumarin) separated from plant extract (Gelsemium sempervirens) were demonstrated earlier from our in vitro studies. In the present study, its in vivo effects have been evaluated in mice. METHODS: Mice were chronically administered 7,12-dimethylbenz [a] anthracene (DMBA) once a week and croton oil twice a week on their back, which resulted in the development of fully grown finger-like projections (papilloma) after 24 weeks. Two subgroups of mice (drug-treated) were treated with two doses of scopoletin (50 mg and 100 mg/kg body weight) respectively while control received 2% ethyl alcohol (the "vehicle" of scopoletin). After the 24-week drug administration, expressions of several key receptors such as aryl hydrocarbon receptor (AhR) and signal proteins like p53, cytochrome P450 1A1 (CYP1A1), proliferating cell nuclear antigen (PCNA), signal transducer and activator of transcription-3 (Stat-3), survivin, matrix metalloproteinase-2 (MMP-2), cyclin D1, c-myc, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and caspase-3, and some anti-oxidant markers were studied. Lipid peroxidation, superoxide dismutase, catalase, glutathione peroxidase and glutathione-s-transferase in supernatant were also detected. RESULTS: Carcinogens induced toxicity, and over-expression of AhR, CYP1A1, PCNA, Stat-3, survivin, MMP-2, cyclin D1 and c-myc and down-regulation of p53, caspase-3 and TIMP-2. In mice treated with scopoletin, the expressions of these proteins and toxicity biomarkers were reverted. CONCLUSION: Since AhR is known to be ligand-activated by DMBA to release signals for several downstream proteins initiating reactive oxygen species generation, the down-regulation of AhR by scopoletin appeared to play a significant role in subsequent down-regulation of some key signal proteins. One possible mechanism of down-regulation of AhR may be through competitive inhibition by scopoletin. Mitogen-activated protein kinases may also have some critical role. This compound can be considered as a possible candidate for chemoprevention.

Encapsulated plant extract (Gelsemium sempervirens) poly (lactide-co-glycolide) nanoparticles enhance cellular uptake and increase bioactivity in vitro.

Ethanolic extract of Gelsemium sempervirens (family: Loganiaceae), henceforth to be called EEGS, is used in various traditional systems of medicine. In homeopathy, EEGS is known as mother tincture of G. sempervirens, which is generally used to treat pain and respiratory ailments. We demonstrated earlier anticancer activity of crude EEGS by in vitro studies on human HeLa cells. To test the hypothesis if nanoparticle-encapsulated extract (now onwards to be called NEEGS) could enhance cellular uptake and thereby improve bioactivity, we formulated nanoparticle encapsulation based on poly (lactide-co-glycolide) (PLGA) and confirmed encapsulation by scanning electron microscopy (SEM) and atomic force microscopy. EEGS was encapsulated with 81.6% efficiency in PLGA biodegradable nanoparticle formulation and F68 (polyoxyethylene-polyoxypropylene) was used as a stabilizer. Dynamic laser light scattering and SEM indicated a particle diameter of 122.6 nm. The zeta potential of the drug-loaded nanoparticles was -14.8 mV. NEEGS was characterized for their biological activities in a skin cancer cell line A375 in vitro. NEEGS exhibited relatively rapid (30 min) and more efficient cellular uptake than their un-encapsulated counterpart (45 min). Analysis of data of apoptosis study using Annexin V-FITC, terminal transferase dUTP nick end labeling assay and DNA ladder revealed that encapsulated EEGS was more potent than their un-encapsulated counterpart in inducing apoptosis of A375 cells. Reverse transcriptase-polymerase chain reaction data of survivin, cyclin-D1, caspase-3, PCNA and p53 also corroborated well to suggest greater potentials of NEEGS as anticancer agents.

Lycopodine from Lycopodium clavatum extract inhibits proliferation of HeLa cells through induction of apoptosis via caspase-3 activation.

Crude ethanolic extract of the plant Lycopodium clavatum has long been used in complementary and alternative medicine for treating various liver ailments and Alzheimer's disease. It has also been claimed to have potential anti-cancer properties in vivo in mice chronically fed liver carcinogens, p-dimethylamino azobenzene (initiator) and phenobarbital (promoter). Incidentally, crude ethanolic extract of Lycopodium clavatum is a mixture of some 201 alkaloids. In order to ascertain if any major fraction can be attributed to have pronounced anti-cancer effect, we examined this major fraction by eluting the crude extract in petroleum ether:ethyl aetate (17:3 vol/vol;) solvent and tried to understand its underlying mechanism. Studies on morphological changes, cell viability and cytotoxicity by microscopy and FACS, Western blot and immunofluorescence of Bcl-2, Bax, cytochrome c, caspase-3 were conducted. Lycopodine was found to induce chromatin condensation, inter-nucleosomal DNA fragmentation and enhanced cell population in sub-G1 region along with increase in reactive oxygen species generation and mitochondrial membrane potential depolarization, release of cytochrome c and activation of caspase-3 which are the events closely involved in apoptosis. An overall analysis of results showed that Lycopodine considerably inhibited growth of HeLa cells which indicates its potential use in chemotherapy.

WITHDRAWN: Anti-cancer potentials of a plant alkaloid (Scopoletin): Induction of apoptosis through caspase activation and cell cycle arrest.

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In vitro studies demonstrate anticancer activity of an alkaloid of the plant Gelsemium sempervirens.

The chemical structure of the main fluorescenting compound in the ethanolic extract (mother tincture) of the American yellow jasmine, Gelsemium sempervirens, was determined by employing (1)H nuclear magnetic resonance (NMR), (13)C NMR, mass spectroscopy, high-performance liquid chromatography (HPLC), correlation spectroscopy (COSY), and Fourier transform infrared (FTIR) spectroscopy analyses. Spectrofluorometric analysis has been made of the mother tincture and its agitated serial dilutions (up to 12th potency) prepared according to a homeopathic procedure in which serial, agitated dilutions were made separately in glass and polypropylene containers. The succussions were made by employing three different modes: hand jerk, sonication, and vortexing. The chemical formula of scopoletin, the main fluorescent compound, was determined to be C(10)H(8)O(4) having a molecular weight of 192.17. Significant differences were noted between the remedies prepared in the two types of containers. Further, a comparison between any two methods of agitation revealed significant differences in fluorometric data of remedies at certain potency levels. The biological (anticancer) action of the crude extract, the alkaloid scopoletin, and 2C potency of Gelsemium sp were tested in vitro on the HeLa cell line through fluorescence microscopy, the 3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, and fluorescent activated cell sorting (FACS). The role of nanoparticles presumably derived from the containers, their orientation, and their interaction with the starting substance during the dynamization process initiated by different modes of agitation could possibly be attributed to the differences noted in the fluorometric data of potencies prepared in the two types of containers and among the three different means of succussion tested.

Comparative efficacy of two microdoses of a potentized homeopathic drug, arsenicum album, to ameliorate toxicity induced by repeated sublethal injections of arsenic trioxide in mice.

OBJECTIVES: To evaluate the efficacy of 2 potentized homeopathic remedies of Arsenicum Album (Ars Alb)--6C and 30C--in combating chronic arsenic toxicity induced by repeated sublethal injections in mice (Mus musculus). METHODS: Mice were randomized and divided into sets: (1) normal (control 1); (2) normal + succussed alcohol (control 2); (3) As(2)O(3) (0.016%) injected at 1 ml/100 g body weight every 7 days (treated); (4) As(2)O(3) injected + succussed alcohol (positive control); (5) As(2)O(3) injected + Ars Alb 6C (drug-fed); (6) As(2)O(3) injected + Ars Alb 30C (drug-fed). Cytogenetical endpoints like chromosome aberrations, micronuclei, mitotic index, sperm head abnormality and biochemical protocols like acid and alkaline phosphatases, aspartate and alanine aminotransferases, reduced glutathione, lipid peroxidation, catalase and succinate dehydrogenase were studied at 30, 60, 90 and 120 days. RESULTS: Compared to controls, chromosome aberrations, micronuclei, sperm head abnormality frequencies and activities of acid and alkaline phosphatases, aspartate and alanine aminotransferases and lipid peroxidation were reduced in both drug-fed series, while mitotic index and activities of glutathione, catalase and succinate dehydrogenase were increased. Ars Alb 30C showed marginally better efficacy than Ars Alb 6C. CONCLUSION: Both remedies indicated potentials of use against arsenic intoxication.