Efficacy of ethanolic spore extract of Lycopodium clavatum in reducing induced hepatotoxicity and genotoxicity in mice

Ethanolic extract of spores of Lycopodium clavatum L., reportedly has profound effect against liver disorders, but lacks adequate experimental validation. To test this claim, healthy inbred Swiss albino mice, Mus musculus, were divided into different groups: Gr.I mice were fed normal diet (negative control); Gr.II - fed normal diet plus ethanol; Gr.III - fed two carcinogens of liver, [0.06% p-dimethyl aminoazobenzene (initiator) and 0.05% phenobarbital (promoter)] known to induce hepatotoxicity and genotoxicity; Gr.IV- mice fed ethanol plus both the carcinogens, and Gr.V- fed carcinogens plus spore extract of Lycopodium clavatum. They were sacrificed at day 90 and 120 for histological studies of liver, assay of cytotoxicity markers and assessment of genotoxicity using endpoints such as chromosome aberrations, micronuclei, mitotic index in bone marrow cells and sperm head anomaly. Additionally, western blot for p53 protein expression and matrix metalloproteinase (MMP) activity in liver was compared among different groups of treated and control mice to evaluate its therapeutic potentials. Compared to Gr.III and IV, less number of mice developed tumors in Gr.V along with significant reduction in hepatotoxicity and genotoxicity, thereby validating its potential use against liver ailments as a herbal remedy.Keywords: p-dimethyl aminoazobenzene, phenobarbital, plant extract, amelioration

Effect of a homeopathic drug, Chelidonium, in amelioration of p-DAB induced hepatocarcinogenesis in mice

BACKGROUND: Crude extracts of Chelidonium majus, and also purified compounds derived from crude extracts of this plant, have been reported to exhibit anti-viral, anti-inflammatory, anti-tumor and anti-microbial properties both in vitro and in vivo. Chelidonium is a homeopathic drug routinely used against various liver disorders including cancer in humans. Two potencies of Chelidonium (Ch-30, Ch-200) have been tested for their possible anti-tumor and enzyme modulating activities in liver and anti-clastogenic effects during p-DAB-induced hepatocarcinogenesis in mice compared to suitable controls. METHODS: Several cytogenetic and enzymatic protocols were used at three fixation intervals; at 60 days, 90 days and 120 days of treatment. Different sets of healthy mice were fed: i) hepatocarcinogen, p-DAB plus phenobarbital (PB), ii) only PB, iii) neither p-DAB nor PB (normal control). One set of mice fed with p-DAB plus PB was also fed Ch-30 (iv) and another set Ch-200 (v). All standard currently used methods were adopted for cytogenetical preparations and for the enzyme assays. RESULTS: All group (i) mice developed tumors in liver at all fixation intervals, while none of group (ii) and (iii) mice developed any tumors. About 40% mice in group (iv) and group (v) did not show tumor nodules in their liver. Feeding of Chelidonium to group (iv) and (v) mice reduced genotoxic effects to a significant extent (p < 0.05 to p < 0.001). CONCLUSION: The homeopathic drug Chelidonium exhibited anti-tumor and anti-genotoxic activities and also favorably modulated activities of some marker enzymes. Microdoses of Chelidonium may be effectively used in combating liver cancer

Ameliorating effect of microdoses of a potentized homeopathic drug, Arsenicum Album, on arsenic-induced toxicity in mice

BACKGROUND: Arsenic in groundwater and its accumulation in plants and animals have assumed a menacing proportion in a large part of West Bengal, India and adjoining areas of Bangladesh. Because of the tremendous magnitude of the problem, there seems to be no way to tackle the problem overnight. Efforts to provide arsenic free water to the millions of people living in these dreaded zones are being made, but are awfully inadequate. In our quest for finding out an easy, safe and affordable means to combat this problem, a homeopathic drug, Arsenicum Album-30, appears to yield promising results in mice. The relative efficacies of two micro doses of this drug, namely, Arsenicum Album-30 and Arsenicum Album-200, in combating arsenic toxicity have been determined in the present study on the basis of some accepted biochemical protocols. METHODS: Mice were divided into different sets of control (both positive and negative) and treated series (As-intoxicated, As-intoxicated plus drug-fed). Alanine amino transferase (ALT) and aspartate amino transferase (AST) activities and reduced glutathione (GSH) level in liver and blood were analyzed in the different series of mice at six different fixation intervals. RESULTS: Both Arsenicum Album-30 and Arsenicum Album-200 ameliorated arsenic-induced toxicity to a considerable extent as compared to various controls. CONCLUSIONS: The results lend further support to our earlier views that microdoses of potentized Arsenicum Album are capable of combating arsenic intoxication in mice, and thus are strong candidates for possible use in human subjects in arsenic contaminated areas under medical supervision

Amelioration of Carcinogen-Induced Toxicity in Mice by Administration of a Potentized Homeopathic Drug, Natrum Sulphuricum 200

To examine if a potentized homeopathic drug, Natrum Sulphuricum 200 (Nat Sulph-200) has protective potentials against hepatocarcinogenesis, liver tumors were induced in mice through chronic feeding of P-dimethylaminoazobenzene (p-DAB; initiator of hepatocarcinogenesis) and phenobarbital (PB; promoter). Mice were divided into five sub-groups: fed normal low protein diet (Gr. I, normal control); fed normal low protein plus alcohol-200 (vehicle of the homeopathic remedy) (Gr. II); fed diet mixed with 0.06% p-DAB plus 0.05% PB (Gr. III); fed diet and carcinogens like Gr.III, plus alcohol 200 (positive control for drug fed mice) (Gr. IV) and fed diet and carcinogens like Gr. III, plus Natrum Sulphuiricum-200 (Gr. V; drug fed). Mice were sacrificed at day 7, 15, 30, 60, 90 and day 120 for study of cytogenetical endpoints like chromosome aberrations (CA), micronuclei (MN), mitotic index (MI) and sperm head anomaly (SHA) and biochemical toxicity parameters like aspartate amino transferase (AST), alanine amino transferase (ALT), acid (AcP) and alkaline (AlkP) phosphatases, lipid peroxidation (LPO) and reduced glutathione (GSH) content. Less number of liver tumors were observed in Gr. V (drug fed) mice. Administration of Nat Sulph 200 reduced genomic damage, activities of AcP, AlkP, AST, ALT, LPO and increased GSH content. Therefore, independent replication of the study by others is encouraged

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

Apigenin, a Bioactive Flavonoid from Lycopodium clavatum, Stimulates Nucleotide Excision Repair Genes to Protect Skin Keratinocytes from Ultraviolet B-Induced Reactive Oxygen Species and DNA Damage

AbstractIn this study, we examined the antioxidative and the DNA protective potentials of apigenin, a flavonoid polyphenol isolated from Lycopodium clavatum, in both in-vitro (HaCaT skin keratinocytes) and in-vivo (mice) models against UV-B radiation. We used DAPI staining in UV-B-irradiated HaCaT skin keratinocytes pre-treated with and without apigenin to assess DNA damage. We also used a flow-cytometric analysis in mice exposed to UV-B radiation with or without topical application of apigenin to assess, through a comet assay, chromosomal aberrations and quanta from reactive oxygen species (ROS) generation. Data from the stability curves for the Gibb's free energy determined from a melting-temperature profile study indicated that apigenin increased the stability of calf thymus DNA. Immunofluorescence studies revealed that apigenin caused a reduction in the number of cyclobutane pyrimidine dimers (CPDs) after 24 h, the time at which the nucleotide excision repair (NER) genes were activated. Thus, apigenin accelerated reversal of UV-B-induced CPDs through up-regulation of NER genes, removal of cyclobutane rings, inhibition of ROS generation, and down-regulation of NF-κB and MAPK, thereby revealing the precise mechanism of DNA repair

Evidences of Protective Potentials of Microdoses of Ultra-High Diluted Arsenic Trioxide in Mice Receiving Repeated Injections of Arsenic Trioxide

The present study was undertaken to examine if microdoses of ultra-high diluted arsenic trioxide (a potentized homeopathic remedy, Arsenicum Album 200C, diluted 10−400 times) have hepatoprotective potentials in mice subjected to repeated injections of arsenic trioxide. Arsenic intoxicated mice were divided into: (i) those receiving Arsenicum Album-200C daily, (ii) those receiving the same dose of diluted succussed alcohol (Alc 200C) and (iii) another group receiving neither drug nor succussed alcohol. Two other control groups were also maintained: one fed normal diet only and the other receiving normal diet and Alc-200C. Toxicity biomarkers like aspartate and alanine aminotransferases, glutathione reductase, catalase, succinate dehydrogenase, superoxide dismutase and reduced glutathione contents were periodically assayed keeping the observer “blinded”. Additionally, electron microscopic studies and gelatin zymography for matrix metalloproteinases of liver tissues were made at day 90 and 120. Blood glucose, hemoglobin, estradiol and testosterone contents were also studied. Compared to controls, Arsenicum Album-200C fed mice showed positive modulations of all parameters studied, thereby providing evidence of protective potentials of the homeopathic drug against chronic arsenic poisoning

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 1060 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

AbstractPolygala 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 β-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

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 C10H16O 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