Evaluation of cytotoxic activity of platinum nanoparticles against normal and cancer cells and its anticancer potential through induction of apoptosis.

BACKGROUND: Plant mediated green synthesis of nanoparticles is an eco-friendly and efficacious approach which finds immense application in the field of medicine. This study aimed to evaluate the cytotoxicity of platinum nanoparticles (ptNPs) synthesized through green technology against normal and different cancer cell lines. METHODS: Platinum nanoparticles were synthesized by green technology and characterized earlier. In this study we examined the cytotoxic effect of platinum nanoparticles (ptNPs) on human lung adenocarcinoma (A549), ovarian teratocarcinoma (PA-1), pancreatic cancer (Mia-Pa-Ca-2) cells and normal peripheral blood mononucleocyte (PBMC) cells and evaluate anticancer potential through induction of apoptosis on PA-1 cells if any. Cytotoxicity was evaluated using MTT assay, trypan blue dye exclusion assay and anticancer potential assessed through clonogenic assay, apoptosis assay, cell cycle analysis. RESULTS: We found that ptNPs exerted cytotoxic effect on cancer cell lines, whereas no cytotoxic effect was observed at highest dose on normal cells. The results showed that ptNPs had potent anticancer activities against PA-1 cell line via induction of apoptosis and cell cycle arrest. CONCLUSION: Overall, these findings have proved that biosynthesized ptNPs could be potent anti-ovarian cancer drugs. Further studies are required to elucidate the molecular mechanism of ptNPs induced anti-tumor effect in vivo.

Biosynthesized Platinum Nanoparticles Inhibit the Proliferation of Human Lung-Cancer Cells in vitro and Delay the Growth of a Human Lung-Tumor Xenograft in vivo: -In vitro and in vivo Anticancer Activity of bio-Pt NPs.

OBJECTIVES: Lung cancer remains a deadly disease with unsatisfactory overall survival. Cisplatin, a standard platinum (Pt)-based chemotherapeutic agent, has the potential to inhibit the growth of lung cancer. Its use, however, is occasionally limited by severe organ toxicity. However, until now, no systematic study has been conducted to verify its efficacy with proper experimental support in vivo. Therefore, we examined whether biosynthesized Pt nanoparticles (NPs) inhibited human lung cancer in vitro and in vivo to validate their use in alternative and complementary medicine. METHODS: We evaluated the in vitro and the in vivo anticancer efficiencies of biosynthesized Pt NPs in a subcutaneous xenograft model with A549 cells. Severe combined immune deficient mice (SCID) were divided into four groups: group 1 being the vehicle control group and groups 2, 3 and 4 being the experimental groups. Once the tumor volume had reached 70 ─ 75 mm(3), the progression profile of the tumor growth kinetics and the body weights of the mice were measured every week for 6 weeks after oral administration of Pt NPs. Doses of Pt NPs of 500, 1,000 and 2,000 mg/kg of body weight were administered to the experimental groups and a dose of honey was administered to the vehicle control group. The efficacy was quantified by using the delay in tumor growth following the administration of Pt NPs of A549 human-lung-cancer xenografts growing in SCID mice. RESULTS: The in vitro cytotoxicity evaluation indicated that Pt NPs, in a dose-dependent manner, inhibited the growth of A549 cells, and the in vivo evaluation showed that Pt NPs at the mid and high doses effectively inhibited and delayed the growth of lung cancer in SCID mice. CONCLUSION: These findings confirm the antitumor properties of biosynthesized Pt NPs and suggest that they may be a cost-effective alternative for the treatment of patients with lung 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.

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.

Protective potentials of a plant extract (Lycopodium clavatum) on mice chronically fed hepato-carcinogens.

Chronic feeding of carcinogens p-dimethylamino azobenzene (initiator) and phenobarbital (promoter) for 90 and 120 days elevated activities of acid and alkaline phosphatases, levels of blood glucose and cortisol and decreased the activities of glutathione reductase, succinate dehydrogenase, and blood cholesterol and hemoglobin contents, and levels of serum estradiol and testosterone in mice. Levels of these biomarkers in both liver and spleen tissues were positively altered along with a significant reduction of tumor incidence in liver of carcinogen intoxicated mice treated with spore extract of Lycopodium clavatum. The results validate the use of this plant extract in complementary and alternative medicines against hepato-toxicity.

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

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A synthetic coumarin (4-methyl-7 hydroxy coumarin) has anti-cancer potentials against DMBA-induced skin cancer in mice.

Scopoletin, an alkaloid separated from ethanolic extract of the medicinal plant, Gelsemium sempervirens (Fam: Loganiaceae) has been reported to have anti-cancer potentials. The synthetic coumarin (4-Methyl-7 hydroxy coumarin) derived from resorcinol and ethyl aceto-acetate in presence of concentrated sulphuric acid is structurally close to scopoletin, being a coumarin derivative. Whether this synthetic compound also has anti-cancer potentials has been evaluated in vivo on DMBA (7,12-Dimethylbenz[a]anthracene) induced skin cancer in mice by analyzing results of several cytogenetic endpoints, Comet assay, and fluorescence activated cell sorting (FACS). Further, expressions of signal proteins like Aryl hydrocarbon receptor , p53, PCNA, Akt, Bcl-2, Bcl-xL, Bad, Bax, NF-kappaB Apaf, IL-6, Cytochrome-c, Caspase-3 and Caspase-9 were studied by immunoblot analysis along with histology of skin and immuno-histochemical localization of Aryl hydrocarbon receptor and PCNA in DMBA treated mice vis-a-vis carcinogen treated synthetic coumarin fed mice. Feeding of this synthetic coumarin induced positive modulations in expression of all biomarkers in DMBA administered mice, giving clues on its possible signaling pathway(s) - primarily through down-regulation of Aryl hydrocarbon receptor and PCNA and up-regulation of apoptotic proteins like Bax, Bad, Cytochrome c, Apaf, Caspase-3 and Caspase-9, resulting in an appreciable reduction in growth of papilloma in mice. Therefore, this synthetic coumarin shows promise for use in cancer therapy, particularly in skin cancer.

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.

Supportive evidence for the anticancerous potential of alternative medicine against hepatocarcinogenesis in mice.

INTRODUCTION: The present study examines if Lycopodium 200 (Lyco-200) has demonstrable anti-cancer activities in mice which are chronically fed carcinogens, p-dimethylaminoazobenzene (p-DAB) and phenobarbital (PB) to induce liver cancer. MATERIALS AND METHODS: Mice in 5 different groups were chronically fed for varying periods of time: group I: normal diet; group II: normal diet + alcohol 200); group III: p-DAB + PB; group IV: p-DAB + PB + alcohol 200 (vehicle of Lyco-200 being ethyl alcohol); group V: p-DAB + PB + Lyco-200. They were sacrificed at day 7, 15, 30, 60, 90 or 120, and the following parameters were assessed: cytogenetic endpoints like chromosome aberrations, micronuclei, mitotic index and sperm-head anomaly; toxicity biomarkers like acid and alkaline phosphatases, alanine and aspartate amino transferase, glutathione reductase, succinate dehydrogenase and catalase activities, lipid peroxidation and reduced glutathione content. Additionally, scanning and transmission electron microscopic analyses of liver tissues were made at day 90 and 120, and immunodetection of p53 protein as well as gelatin zymography for matrix metalloproteinases in liver tissue were performed. Furthermore, studies were conducted on blood glucose, hemoglobin and cholesterol, estradiol, testosterone and cortisol, and lymphocyte and hepatic cell viabilities. Physical properties of Lyco-200 and potentized alcohol 200 were analyzed by using methods such as UV, Fourier Transform Infrared Spectroscopy (FTIR), Fluorescence Spectroscopy, 1H-NMR and 13C-NMR (Nuclear Magnetic Resonance Spectroscopy). RESULTS: Lyco-200 reduced cytogenetic damages yielding positive modulations of all biochemical, pathological and other risk factors, cell viability and expression of p53 protein and matrix metalloproteinases as compared to controls. CONCLUSION: Studies on other mammals are recommended to further investigate the potential of Lyco-200 in liver cancer.