Regulation of Caspase-3 and Bcl-2 Expression in Dalton's Lymphoma Ascites Cells by Abrin.

The role of abrin, a toxic lectin isolated from seeds of Abrus precatorius Linn in inducing apoptosis in murine Dalton's Lymphoma Ascites (DLA) cells was evaluated. Abrin when incubated at the concentration of 10 ng per million DLA cells could bring about cell death as typical morphological changes with apoptosis. However, necrotic cell death dominated when a higher dose of abrin was used. DNA samples, isolated from DLA cells treated with abrin showed fragmentation. Abrin brought about induction of apoptosis by stimulating the expression of pro-apoptotic Caspase-3, at the same time blocking the expression of Bcl-2, which is an anti apoptotic gene. However, the expression of tumor suppressor gene p53 has not been observed in control and abrin-treated DLA cells. Results suggested that abrin effectively induced apoptotic changes in the tumor cells that led to cellular death.

Effect of Smoke Shield-a herbal formulation on the mutagenicity and oxidative stress produced by cigarette smoke in rats.

Herbal formulation Smoke Shield was studied for its activity against cigarette smoke induced oxidative stress in rats. Smoke Shield administration was found to decrease the increased lipid peroxidation and conjugated dienes in the lung tissue and serum of cigarette smoke exposed rats. Moreover, administration of Smoke Shield was found to increase the activity of catalase in erythrocytes and lung tissue of rats, which was decreased upon smoke exposure. Glutathione peroxidase, which was increased by smoke exposure, was decreased by Smoke Shield and concomitantly there were increased levels of glutathione in the cells. In vitro studies indicated that addition of Smoke Shield could reduce the mutagenicity produced by tobacco smoke condensate, mosquito coil smoke condensate as well as by aqueous tobacco extract. In vitro antioxidant potential of smoke shield was found to be comparable or better than known antioxidants such as vitamin C and vitamin E. These results indicated that Smoke Shield administration could effectively reduce the oxidative stress produced in rats by cigarette smoke and it could inhibit mutagenicity produced by smoke condensates and tobacco extract in Salmonella typhimurium.

Chemoprotective activity of an extract of Phyllanthus amarus against cyclophosphamide induced toxicity in mice.

The effect of 75% methanolic extract of the plant Phyllanthus amarus (P. amarus) was studied against cyclophosphamide (CTX) induced toxicity in mice. Administration of CTX (25 mg/kg b.wt, i.p.) for 14 days produced significant myelosuppression as seen from the decreased WBC count and bone marrow cellularity. Administration of P. amarus extract at doses 250 and 750 mg/kg b.wt significantly reduced the myelosuppression and improved the WBC count, bone marrow cellularity as well as the number of maturing monocytes. CTX treatment also reduced the activity of glutathione system and increased the activity of phase I enzyme that metabolize CTX to its toxic side products. P. amarus administration was found to decrease the activity of phase I enzyme. Administration of P. amarus also increased the cellular glutathione (GSH) and glutathione-S-transferase (GST), thereby decreasing the effect of toxic metabolites of CTX on the cells. Administration of P. amarus did not reduce the tumor reducing activity of CTX. In fact, there was a synergistic action of CTX and P. amarus in reducing the solid tumors in mice. Results indicated that administration of P. amarus can significantly reduce the toxic side effects of CTX and is not interfering with the antitumor efficiency of CTX.

Antimutagenicity of herbal detoxification formula Smoke Shield against environmental mutagens.

Smoke Shield is a formulation designed to reduce smoke related mutagenicity and toxicity in the population. Smoke Shield contains a dual extract of turmeric (Curcuma longa) obtained by supercritical CO2 gas extraction and post-supercritical hydroethanolic extraction together with extracts of green tea and other spices, whose presence synergistically increases the activity of turmeric. In the present study we have shown its antimutagenic activity to various environmental mutagens in vitro and in vivo. Smoke Shield was found to produce significant inhibition of mutagenicity to Salmonella typhimurium induced by sodium azide and 4-nitro-0-phenylenediamine (NPD) at a concentration of 2 mg/plate while inhibition to N-methyl-N-nitro N'nitrosoguanidine was less significant. Inhibition was also found to depend upon the strain which was used. Smoke Shield was found to be more effective against mutagens needing metabolic activation such as 2-Acetamidofluorene (2-AAF) and benzo[a]pyrene. Smoke Shield was also found to significantly inhibit the mutagenicity induced by tobacco extract to Salmonella typhimurium TA102. Smoke Shield was also found to inhibit the urinary mutagenicity of rats treated with the benzo[a]pyrene and tobacco extract. Moreover, Smoke Shield administration was found to inhibit the urinary mutagenicity in smokers. These results indicate that Smoke Shield could inhibit mutagenic response in vitro and in vivo produced by several kinds of mutagens present in our atmosphere.

Antioxidant activity of brahma rasayana.

Free oxygen radical scavenging activity of brahma rasayana (BR) was studied by in vitro and in vivo models. Addition of aqueous extract of BR was found to scavenge the lipid peroxides already present in rat liver homogenate (IC50 700 micrograms/ml) and inhibit the lipid peroxide generated by Fe(2+)-ascorbate (IC50 2600 micrograms/ml) and Fe(3+)-ADP-ascorbate system (IC50 1200 micrograms/ml). BR was found to scavenge the hydroxyl radical generated by Fenton reaction (IC50 7400 micrograms/ml) and superoxide generated by photoreduction of riboflavin (IC50 180 micrograms/ml). BR was also found to inhibit the nitric oxide radical generated in vitro from sodium nitroprusside (IC50 5.5 micrograms/ml). Oral administration of BR (50 mg/dose/animal) was found to inhibit the PMA induced superoxide generation in mice peritoneal macrophages. Oral administration of BR; 10 and 50 mg/dose/animal was also found to inhibit the nitrite production in peritoneal macrophages and percentage inhibition was 25.2% and 37.8% respectively. These results indicate significant antioxidant activity of BR in vitro and in vivo.

Effect of Brahma Rasayana on antioxidant systems and cytokine levels in mice during cyclophosphamide administration.

Intraperitoneal administration of cyclophosphamide (CTX) 25 mg/kg.b.wt. dose/mouse for 10 days was found to suppress the tissue and serum level of reduced glutathione (GSH), blood glutathione peroxidase (GPX) and tissue levels of superoxide dismutase (SOD) and catalase (CAT). Tissue levels of glutathione reductase (GR) and glutathione-S-transferase (GST) were unaltered by CTX treatment while serum and tissue lipid peroxide levels were significantly increased. Oral administration of Brahma Rasayana BR-50 mg/dose/mouse for 10 days and 30 days significantly enhanced the tissue levels of SOD, CAT, GST, GPX, serum and tissue GSH and significantly reduced the serum and tissue lipid peroxidation. BR treatment was also found to enhance the serum cytokine level of interferon-gamma (IFN-gamma), interleukin-2 (IL-2) and granulocyte macrophage-colony stimulating factor (GM-CSF) in normal and CTX treated mice. The results are indicative of the use of BR to reduce the oxidant stress induced by CTX treatment and its effect in cellular function.

Protective effect of Picroliv, the active constituent of Picrorhiza kurroa, against chemical carcinogenesis in mice.

Cancer chemoprevention of chemically induced tumours by Picroliv, an iridoid glycoside mixture purified from Picrorhiza kurroa, was studied on 20-methylcholanthrene (20-MC)-induced sarcoma model and 7,12-dimethylbenz[a]anthracene (DMBA)-initiated papilloma formation in BALB/c mice. Administration of Picroliv (100 and 200 mg/kg, p.o) inhibited the sarcoma development by 47 and 53% as estimated on day 200 after 20-MC administration. Control animals started dying of tumour burden 76 days after 20-MC administration and all animals were dead by day 170, while 60 and 66% of the animals survived in the Picroliv treated group, 100 and 200 mg/kg, respectively. Picroliv exhibited anti-tumour-promoting activity on a two-stage carcinogenesis test on mouse skin using DMBA as an initiator and croton oil as a promoter. Topical application of Picroliv (1 and 5 mg/mouse) 30 minutes prior to that of croton oil application resulted in a 50 and 60% reduction in the number of animals that developed papillomas, and 48 and 64% reduction in the number of papillomas per mouse. There was also a delay in the onset of first skin tumour in the group of animals treated with Picroliv. Oral administration of Picroliv (150 mg/kg, p.o.) prior to DMBA application delayed the onset of papillomas and the percent of mice (60%) with tumours indicates that Picroliv inhibited the tumour initiation induced by DMBA. Picroliv administration was also found to increase the life span of transplanted Dalton's Lymphoma Ascites (DLA) and Ehrlich Ascites Carcinoma (EAC) harboring mice and reduced the volume of transplanted solid tumours.

Inhibition of chemical carcinogenesis by berberine in rats and mice.

Berberine, an alkaloid isolated from the plant Berberis aristata, has been found to inhibit significantly the carcinogenesis induced by 20-methylcholanthrene (200 microg/0.1 mL/mouse) or N-nitrosodiethylamine (NDEA; 0.02% NDEA in distilled water, 2.5 mL/animal by gavage, five days a week for 20 weeks) in a dose-dependent manner in small animals. Administration of berberine (0.5, 2.5 or 5.0 mg kg(-1)) could reduce significantly the incidence of tumour in animals after an injection of 20-methylcholanthrene and increased their life span compared with the control. When berberine (10, 25 or 50 mg kg(-1)) was administered simultaneously with NDEA, the markers of liver injury (liver weight, gamma-glutamyl transpeptidase activity and glutathione S-transferase level) were reduced significantly compared with animals treated with NDEA only, which resulted in all the values being elevated. A similar decrease was noted in the serum levels of lipid peroxide, bilirubin and glutamate pyruvate transaminase. Morphology of liver tissue and levels of marker enzymes indicated that berberine offered protection against chemical carcinogenesis.

Antitumour activity of Emblica officinalis.

Aqueous extract of Emblica officinalis (E.O) was found to be cytotoxic to L 929 cells in culture in a dose dependent manner. Concentration needed for 50% inhibition was found to be 16.5 microg/ml. E.O and chyavanaprash (a non-toxic herbal preparation containing 50% E.O) extracts were found to reduce ascites and solid tumours in mice induced by DLA cells. Animals treated with 1.25 g/kg b.wt. of E.O extract increased life span of tumour bearing animals (20%) while animals treated with 2.5 g/kg b.wt. of chyavanaprash produced 60.9% increased in the life span. Both E.O and chyavanaprash significantly reduced the solid tumours. Tumour volume of control animals on 30th day was 4.6 ml where as animals treated with 1.25 g/Kg b.wt. of E.O extract and 2.5 g/kg b.wt. of chyavanaprash showed a tumour volume of 1.75 and 0.75 ml, respectively. E.O extract was found to inhibit cell cycle regulating enzymes cdc 25 phosphatase in a dose dependent manner. Concentration needed for 50% inhibition of cdc 25 phosphatase was found to be 5 microg/ml and that needed for inhibition of cdc2 kinase was found to be >100 microg/ml. The results suggest that antitumour activity of E.O extract may partially be due to its interaction with cell cycle regulation.

Effect of brahma rasayana on antioxidant system after radiation.

Oral administration of brahma rasayana (BR; 50 mg/animal for 10 and 30 days) significantly increased the liver antioxidant enzymes such as superoxide dismutase (SOD), catalase(CAT) and tissue and serum levels of reduced glutathione (GSH). Whole body irradiation suppressed the levels of SOD, CAT and GSH. Reduced activity of SOD, CAT and GSH was significantly elevated by treatment with BR after radiation treatment. Similarly radiation exposure induced increase in serum and liver lipid peroxides was significantly reduced by further treatment with BR. The results indicate that BR could ameliorate the oxidative damage produced in the body by radiation and may be useful as an adjuvant during radiation therapy.

Cancare-a herbal formulation inhibits chemically induced tumours in experimental animals.

Cancer chemopreventive potential of Cancare, a multi-herbal formulation on chemically induced tumours was studied by N-nitrosodiethylamine (NDEA) induced hepatocarcinogenesis in rats and 20-methylcholanthrene (20-MC) induced sarcoma development in mice. Oral administration of Cancare was found to inhibit the liver tumour development induced by N-nitrosodiethylamine. Animals administered with NDEA had visible liver tumours by the end of 30th weeks and the liver weight was raised to 6.1 +/- 1.4 g/ 100 g body wt. None of the animals treated with Cancare (150 mg/ kg) developed any visible liver tumours by this period and the liver weight was 3.0 +/- 0.6 g/ 100 g body wt. Gamma-Glutamyl transpeptidase, a marker of hepatocellularcarcinoma, which was raised to 83.7 +/- 8. 9 U/l in serum of NDEA treated group was reduced to 35.2 +/- 6.1 U/l by simultaneous administration of Cancare. Elevated levels of serum alkaline phosphatase, glutamate pyruvate transaminase, bilirubin, liver glutathione S-transferase, glutathione and gamma-Glutamyl transpeptidase in the NDEA administered group was significantly reduced by Cancare administration. Cancare administration inhibited the sarcoma development and increased the life span of mice administered with 20-MC dose dependently. All animals in the control group developed sarcomas by 150th day and dead by 174th day after 20-MC administration. Cancare administration (30 mg and 150 mg/kg) inhibited the sarcoma development (46.7 and 60%) as well as increased the life span (53.3 and 66.7%) as estimated on 240th day after 20-MC administration. The results are indicative of the chemopreventive potential of Cancare against chemically induced neoplasmas.

Phyllanthus amarus extract administration increases the life span of rats with hepatocellular carcinoma.

The effect of Phyllanthus amarus extract administration after induction of hepatocellular carcinoma (HCC) by N-nitrosodiethylamine (NDEA) was studied in Wistar rats. Administration of an aqueous extract of P. amarus was found to significantly increase the survival of hepatocellular carcinoma harboring animals. All the untreated rats died of tumour burden by 33.7+/-1.6 weeks. Administration of P. amarus extract (150 mg/kg b.w.) after tumour development increased the survival of animals to an average of 52. 2+/-2.3 weeks. Serum gamma-glutamyl transpeptidase activity which was elevated to 182+/-23 U/l by NDEA administration was lowered to 112+/-19 U/l by the administration of P. amarus extract. Similarly elevated glutathione S-transferase activity (1534+/-116 nmol/min per mg protein) and glutathione (20.5+/-2.4 nmol/mg protein) levels in the NDEA administered group were found to be lowered to 1112+/-89 nmol/min per mg protein and 14.2+/-2.2 nmol/mg protein respectively. P. amarus administration was found to be ineffective in controlling the liver weight, elevation of tissue gamma-glutamyl transpeptidase, serum alkaline phosphatase and serum glutamate pyruvate transaminase of HCC harboring animals.

Hepatoprotective activity of Emblica officinalis and Chyavanaprash.

Hepatoprotective activity of Emblica officinalis (EO) and Chyavanaprash (CHY) extracts were studied using carbon tetrachloride (CCl(4)) induced liver injury model in rats. EO and CHY extracts were found to inhibit the hepatotoxicity produced by acute and chronic CCl(4) administration as seen from the decreased levels of serum and liver lipid peroxides (LPO), glutamate-pyruvate transaminase (GPT), and alkaline phosphatase (ALP). Chronic CCl(4) administration was also found to produce liver fibrosis as seen from the increased levels of collagen-hydroxyproline and pathological analysis. EO and CHY extracts were found to reduce these elevated levels significantly, indicating that the extract could inhibit the induction of fibrosis in rats.

Role of natural killer cells in iscador mediated inhibition of metastasis by adoptive immunotherapy.

Iscador activated (in vivo and in vitro) splenocytes were found to inhibit metastatic tumour growth in C57BL/6 mice. In order to check whether NK cells are involved in the antimetastatic activity of Iscador activated splenocytes ,animals were depleted of NK cells using antiasialo GMI antibodies. When spleen cells activated in vivo with Iscador were injected into animals pretreated with Antiasialo GM I antibodies, there was an average of 44.6 tumour nodules on 21st day indicating that stimulation of NK cell activity produced by the Iscador compensate for the NK cell depletion by Antiasialo GM I antibody. Animals treated with Iscador activated splenocytes showed an average survival period of 68 days whereas that of control tumour bearing animals treated with Ab the average survival was 19.3 days. The lung collagen hydroxyproline content, serum sialic acid levels, markers of metastasis were also significantly (P<0.001) lowered in these animals compared to those of the untreated tumour bearing animals. gamma-glutamyl transpeptidase a marker of neoplastic growth, was also significantly reduced (P<0.001) in animals treated with activated splenocytes.

Effect of Picrorrhiza kurroa extract on transplanted tumours and chemical carcinogenesis in mice.

Anti-tumour and anti-carcinogenic activity of Picrorrhiza kurroa extract were studied in mice. Administration of 20-methylcholanthrene (20 MC) produced 100% induction of sarcoma in control mice, whereas the tumour incidence and tumour related deaths were significantly inhibited by the oral administration of P. kurroa extract 150 and 750 mg/kg body weight, respectively. The extract was also found to reduce the volume of transplanted solid tumours induced by Dalton's lymphoma ascites (DLA) tumour cell lines and increased the life span of ascites tumour bearing mice. P. kurroa extract inhibited yeast topoisomerase I and II enzyme activity when tested on Saccharomyces cerevisiae mutant cell cultures. The extract did not inhibit the enzyme involved in the activation of carcinogen and the cell cycle regulatory enzyme cdc2 kinase.

Effect of herbal preparation, brahma rasayana, in amelioration of radiation induced damage.

Oral administration of brahma rasayana (BR; 10 and 50 mg/dose/animal) for 15 days increased significantly total leukocyte count and percentage of polymorphonuclear cells in irradiated mice. Bone marrow cellularity and alpha-esterase positive cells also increased significantly in radiation-treated animals after BR administration. Number of nodular colonies on the surface of spleen on day seven increased significantly in lethally irradiated recipients receiving bone marrow cells from animals treated with BR. Oral administration of BR also enhanced in serum level of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and granulocyte macrophage-colony stimulating factor(GM-CSF) in normal and irradiated mice. These results indicated that proliferation of stem cells induced by BR in irradiated mice may be related to its stimulation of cytokine production.

Inhibition of N-nitrosodiethylamine-induced hepatocarcinogenesis by Picroliv.

Picroliv, an iridoid glycoside mixture prepared from the roots and rhizomes of Picrorhiza kurroa was found to be an effective inhibitor of hepatocarcinogenesis induced by N-Nitrosodiethylamine (NDEA) in rats. Animals administered with NDEA had large hepatic nodules and the liver weight was increased to 6.17 +/- 1.0 g/100 g.b.wt. as compared to the normal liver weight 2.84 +/- 0.08 g/100 g.b.wt. Picroliv administration (200 mg/Kg.b.wt) reduced the liver weight to 3.30 +/- 0.23 g/100 g.b.wt. Oral administration of Picroliv reduced NDEA-induced elevation of gamma-glutamyltranspeptidase (gamma-GT) in serum and liver to that of normal rats. Moreover, elevated levels of bilirubin, alkaline phosphatase (ALP), glutamatepyruvate transaminase (GPT) and serum peroxides were also found to be significantly reduced by Picroliv administration. Similar observations were noticed in glutathione (GSH) and glutathione S-transferase (GST) levels. Histopathological analysis of the Picroliv treated rat liver resembles that of a normal liver except for a few alterations such as hepatocytomegalia and karyomegalia in some focii. The results are indicative of the chemopreventive potential of Picroliv against chemically-induced liver tumours.

Myeloprotective effect of a non-toxic indigenous preparation Rasayana in cancer patients receiving chemotherapy and radiation therapy. A pilot study.

The effect of an indigenous medical preparation--Brahma Rasayana (BR)--on the haemopoetic protection in cancer patients undergoing radio in association with chemotherapy was studied. Administration of BR accelerated the recovery of the haemopoetic system as seen by a rapid rise in total leukocytes. Both lymphocytes and neutrophils were significantly increased by Rasayana treatment. Nadir of WBC was 3633 +/- 120 and 2954 +/- 305 in treated and untreated patients. Nadir of neutrophils was 2830 +/- 964 in treated patients and 1791 +/- 922 in untreated patients. Nadir of lymphocytes remained almost unchanged. Total number of consecutive days of leukopenia, neutropenia and lymphopenia was also significantly reduced after the treatment. Rasayana treatment also made serum lipid peroxidation decrease confirming its capacity of reducing oxidative stress induced by cancer treatment. The use of this non-toxic preparation as an adjuvant in cancer therapy will prove out to be highly beneficial.

Effect of "rasayanas" a herbal drug preparation on cell-mediated immune responses in tumour bearing mice.

Administration of herbal preparation, Rasayanas has been found to enhance the natural killer cell activity in normal as well asin tumour bearing animals. Brahma Rasayana (BR) was found to have the maximum activity. BRand Aswagandha Rasayana (AR) were found to activate antibody-dependent cellular cytotoxicity significantly. AR was also found to activate macrophages. All the Rasayanas were found to stimulate antibody dependent complement mediated tumour cell lysis. The results of these studies indicate usefullness of Rasayanas for immunostimulation in normal and in disease state.

Effect of "rasayanas", a herbal drug preparation on immune responses and its significance in cancer treatment.

Rasayanas are considered to be immunostimulating preparations used extensively in indigenous medical practice. However there are only very few reports to substantiate this claim, and this paper gives preliminary evidence for the potentiation of immunity by Rasayanas given to mice orally. Administration of Rasayanas were found to enhance the proliferation of spleen cells significantly especially in the presence of mitogen. Similar result was also seen with bone marrow cells; however mitogenic stimulation could not be observed. Esterase activity was found to be enhanced in bone marrow cells indicating increased maturation of cells of lymphoid linkage. Rasayanas also enhanced humoral immune response as seen from the increased number of antibody forming cells and circulating antibody titre. These results indicate the usefulness of Rasayana as immunostimulating agent.