Antioxidant and anti-inflammatory activities of the plant Andrographis paniculata Nees

In this study, we explored the antioxidant and anti-inflammatory properties of the medicinal herb Andrographis paniculata using in vitro as well as in vivo systems. Methanolic extract of Andrographis paniculata was found to inhibit formation of oxygen derived free radicals such as superoxide (32%) hydroxyl radicals (80%) lipid peroxidation (80%) and nitric oxide (42.8%) in in vitro system. In vivo studies using BALB/c mice models also showed significant inhibition in PMA induced superoxide (32.4%) and nitric oxide (65.3%) formation. Interestingly we also found that, administration of Andrographis paniculata extract produced complete inhibition of carageenan induced inflammation compared with control models.     

Cytokine profile and natural killer cell activity in Listeria monocytogenes infected mice treated orally with Petiveria alliacea extract

In this work, we investigated the effects of Petiveria alliacea extract on the production of Th1-type and Th2-type cytokines and on NK cells activity in normal and Listeria monocytogenes infected mice. Our results demonstrated that in normal/non-infected mice P. alliacea administration led to increased levels of Interleukin-2 (IL-2). The infection alone enhanced INF-γ levels and NK cell activity at 48 and 72 hours of infection. The treatment with five consecutive doses of 1000mg/kg/day of P. alliacea extract, given previously to infection, led to further increases in IL-2 levels, in relation to normal/non-infected/P. alliacea treated controls, and in INF-γ levels at 72h of infection, compared to infected mice. On the other hand, the production of IL-4 and IL-10 were not altered either by the infection or by the treatment with P. alliacea extract. NK cells activity increased at 48h and 72h following the inoculation of the bacteria. When mice were treated with P. alliacea previously to infection, NK activity was higher than that observed at 48h, 72h and 120h of infection in the infected animal. Based on these findings we suggest that P. alliacea up-regulates anti-bacterial immune response by enhancing both Th1 function and the activity of NK cells.     

ADJUVANT EFFECT OF PLUCHEA QUITOC EXTRACT ON THE RESISTANCE OF TUMORBEARING MICE BY MODULATION OF THE HOST HEMATOPOIETIC RESPONSE

Progressive tumor growth is regularly accompanied by changes in the cellular constituents of the immune system. Evidence suggests that soluble factors generated during tumor growth can affect the amount of granulocytemacrophage progenitors. In vitro colony growth of progenitor cells may be an early indicator of the cellular changes associated with tumor growth. Pluchea quitoc has been previously found to modulate the hematopoietic response during bacterial infection. This study was designed to investigate the effects of P. quitoc on the growth and differentiation of bone marrow granulocyte-macrophage progenitor cells (CFU-GM) in Ehrlich ascites tumor-bearing mice. In contrast to the myelosuppression developed in the tumor-bearing animals, treatment with P. quitoc ethanolic extract (250, 500 or 1000 mg/kg) for 3 consecutive days after tumor challenge reversibly stimulated myelopoiesis, restoring the number of CFU-GM to normal. This same dose-schedule also increased colony formation in normal mice as compared to controls. In addition, P. quitoc significantly enhanced survival of tumor-bearing mice. These results suggest an immunoregulatory role for P. quitoc in counteracting the tumor-induced myelopoietic suppression as well as usefulness as adjuvant treatment of cancer.     

Stimulatory action of Pluchea quitoc extract on the hematopoietic response during murine listeriosis

The importance of both granulocytes and macrophages in the response to Listerin monocytogenes infection make this infection a suitable choice to investigate the effects of Pluchea quitoc on hematopoiesis. A significant depletion of bone marrow granulocyte-macrophage progenitor cells (CFU-GM) was observed at 48 and 72 h after intraperitoneal infection of mice with 1 ± 10⁴ L. monocytogenes. However, the treatment of infected animals with P. quitoc ethanolic extract (250, 500 or 1000 mg/kg) given orally for 3 consecutive days prior to infection produced a stimulatory effect on myelopoiesis, restoring the number of CFU-GM to normal. This same dose-schedule also increased colony formation in normal mice as compared to controls. In addition, P. quitoc significantly enhanced survival of infected mice. Thus, it is probable that the ability of P. quitoc to induce a higher reserve of granulocyte-macrophage precursors in the bone marrow is of major significance in determining early resistance to infection.     

Activation of cytotoxic T lymphocyte responses and attenuation of tumor growth in vivo by Andrographis paniculata extract and andrographolide

The stimulatory effect of Andrographis paniculata extract and andrographolide on cytotoxic T lymphocyte (CTL) production was determined in BALB/c mice by Winn's neutralization assay using CTL sensitive EL4 thymoma cells as target cell. Extract and andrographolide showed a significant increase in CTL production in both the in vivo and in vitro models. The survival time of EL4 cells alone in animals was only 27.1 days and it was increased to 51.1 and 44.5 days in extract- and andrographolide treated animals with percentage increase in life span (%ILS) of 88.5 and 64.2, respectively. The survival rate of animals administered EL4 cells incubated with alloimmunized spleen cells (effector cells) from normal BALB/c mice was 35.8 (%ILS 32.1). When this group was treated with 10 doses of extract and andrographolide the life span was further increased to 52.1 days (%ILS 92.2 ) and 48.1 days (%ILS 77.4). Survival days of animal carrying EL4 cells incubated with alloimmunized spleen cells (effector cells) from extract and andrographolide treated animals were 55.5 and 50.3 days respectively. When these animals continued with extract and andrographolide treatment for 10 days their life spans were significantly increased to 62 and 53.8 days, respectively. The level of cytokines such as Interlevkin (IL)-2 and Interferon (IFN)-γ also was enhanced in these animals when they were treated with extract and andrographolide. This study demonstrated that A. paniculata extract and andrographolide stimulate the CTL production through enhanced secretion of IL-2 and IFN-γ by T cells and thereby inhibit the tumor growth.     

Inhibition of inflammatory angiogenesis in rats by loco-regional administration of hydrocortisone and protamine

We have studied the antiangiogenetic effects of hydrocortisone and protamine given intra-arterially. The cornea of male, Sprague-Dawley rats were cauterized with silver nitrate. The following treatments were given :30 g hydrocortisone topical (t.p.), b.i.d., 50 mg/kg/day intraperitoneally (i.p.) or intra-arterially (i.a.), 10 mg/ kg/day protamine i.p. or i.a. Saline was administered to the control groups. In separate experiments we also evaluated the anti-inflammatory effects of hydrocortisone, i.p., on the cauterized corneas.Five days after cauterization, the animals were killed, exsanguinated and India ink was injected to show the network of neovessels. The percentage area of the cornea covered by neovessels was measured morphometrically and evaluated statistically. Hydrocortisone t.p. (–84%), i.a. (–60%) and protamine i.a. (–44%) significantly inhibited angiogenesis in the cauterized cornea. Either drugs, i.p., had any antiangiogenetic effects, but hydrocortisone significantly reduced cell infiltration of the corneas. The results suggest that locoregional administration of antiangiogenetic drugs might be clinically useful.     

Influence of berberine on T-cell mediated immunity

The protoberberine alkaloid berberine is isolated as a main alkaloid from the roots and bark of Berberis vulgaris. Berberine strongly inhibited in vitro the proliferative response of mouse spleen cells to T-dependent mitogens concanavalin A (Con A) and phytochemagglutinin (PHA). Spleen cells obtained from berberine-treated mice (10 mg/kg/3 days) expressed enhanced proliferative response to both mitogens. Berberine was applied to mice at different intervals before or after the induction of adjuvant arthritis (AIA) and Candida albicans (C. albicans) infection. The application of the alkaloid to new born mice (5 days after birth at a dose of 5 mg/kg/3 days) did not change the course of AIA and C. albicans infection. Its application at three 10 day intervals (5 mg/kg), starting from the 5 day after birth increased the joint inflammation in AIA. The host resistance to C. albicans infection was not affected, while the delayed type hypersensitivity (DTH)-reaction against the pathogen was enhanced. The alkaloid inhibited the development of AIA when applied after its onset (10 mg/kg from day ±3 to ±12 day). Berberine treatment during the ongoing infection did not influence its outcome (from ±2 to ±10 day).     

Modification of clastogenicity of three known clastogens by garlic extract in mice in vivo

The anticlastogenic activity of crude extract of garlic (Allium sativum L.) was studied in bone marrow cells of mice. Male laboratory-bred Swiss albino mice were given one of three concentrations of the freshly prepared extract (100 mg, 50 mg, and 25 mg/kg body weight) as a dietary supplement by gavage for 6 consecutive days. On the seventh day the mice were administered a single acute dose of two known clastogens, mitomycin C(1.5 mg/kg) and cyclophosphamide (25 mg/kg) or sodium arsenite (2.5 mg/kg), simultaneously with garlic extract. After 24 hr, chromosome preparations were made from the bone marrow cells. The endpoint studied were chromosomal aberrations and damaged cells. Garlic extract alone induced a low level of chromosomal damage. The clastogenicity of all three mutagens were reduced significantly in the animals which had been given garlic extract as dietary supplement. The extent of reduction was different for the three clastogens and may be attributed to the interaction with the different components of the extract. © 1993 Wiley-Liss, Inc.     

EVALUATION OF CAESALPINIA FERREA EXTRACT ON BONE MARROW HEMATOPOIESIS IN THE MURINE MODELS OF LISTERIOSIS AND EHRLICH ASCITES TUMOR

The capacity of hematopoietic tissues to produce and mobilize phagocytes to the site of infection and tumor growth is of central importance to mediate the early immunological response. In this perspective, studies from our laboratory have defined Listeria monocytogenes infection and the Ehrlich ascites tumor (EAT) as useful models to investigate the effects of natural compounds on the growth and differentiation of granulocyte-macrophage progenitor cells (CFU-GM). As expected, a significant reduction in the number of bone marrow CFU-GM was observed in the initial stages of infection with a sublethal dose of Listeria. Similarly, the bone marrow CFU-GM decreased sharply 4 days after the EAT transplantation. Treatment of infected and tumor-bearing mice with 500 and 1000 mg/kg of Caesalpinia ferrea aqueous extract, given 3 times orally, significantly stimulated myelopoiesis, whereas no effects were observed with the 250 mg/kg dose. Similar results were obtained in normal mice. The administration of the two higher doses of the extract also protected 15-20% of mice from a lethal dose of Listeria and significantly prolonged survival of EAT-bearing mice. In summary, these results demonstrate that C. ferrea extract acts as a positive regulator of myelopoiesis, and suggest that the therapeutic effect of C. ferrea may be partially mediated by this action.     

Analgesic and Anti-Inflammatory Activities of Asparagus Africanus Root Extract

The methanolic extract of the roots of Asparagus africanus Lam (Liliaceae) which contains mainly saponins and carbohydrate showed significant analgesic and anti-inflammatory activities (P<0.05) in the tail-flick/hot-plate test and egg albumen-induced rat paw oedema tests that were comparable to the test drugs (morphine 20mg/kg and indomethacin 50mg/kg respectively). These results indicate that the extract possesses analgesic and anti-inflammatory properties.     

Ipomoea obscura (L.) enhances the functions of immunological effector cells, inhibits proinflammatory cytokines and nitric oxide production by LPS induced macrophages

Most of the synthetic chemotherapeutic agents available today are immunosuppressant, cytotoxic and exerts variety of side effects. Botanical based immunomodulators are often employed as supportive or adjuvant therapy to overcome the undesired effects of cytotoxic chemotherapeutic agents and to restore normal health. The methanolic extract of traditionally important medicinal plant Ipomoea obscura exhibited immunomodulatory activity in BALB/c mice. Intraperitoneal administration of five doses of the extract (10 mg/kg body wt) was found to enhance the total WBC count (13912 cells/mm(3)) on the 12(th) day, bone marrow cellularity (28.9 x 10(6)cells/femur) and number of alpha-esterase positive cells (1246 cells/4000 cells). Treatment with the extract along with the antigen, sheep red blood cells (SRBC), produced an enhancement in the circulating antibody titer and the number of plaque forming cells (PFC) in the spleen. Maximum number of PFC (267.6 PFC/10(6) spleen cells) was obtained on the 6(th) day. At the same time administration of Ipomoea obscura extract significantly reduced the elevated levels of proinflammatory cytokines and nitric oxide production by lipopolysaccharide stimulated macrophages. These results indicate the immunomodulatory activity of the alcoholic extract of Ipomoea obscura.     

Modulation by Acanthospermum australe extracts of the tumor induced hematopoietic changes in mice

Previous studies on the Ehrlich ascites tumor (EAT) model indicate that tumor progression is associated with reduced myelopoiesis and increased extramedullar hematopoiesis. In order to investigate the in vivo antitumor activity of Acanthospermum australe, its hydroalcoholic extract was partitioned with different solvents and the resulting extracts were monitored by their effects on bone marrow and spleen hematopoietic progenitor cell proliferation and differentiation in EAT-bearing mice. Oral treatment of tumor-bearing mice with 3 doses of 100, 500 and 1000 mg/kg of the crude hydroalcoholic extract and its chloroformic, butanolic and aqueous fractions significantly stimulated myelepoiesis and brought extramedullar hematopoiesis back to near control values. In normal mice, stimulation of myelopoiesis was only observed with the crude and the butanolic extracts. All the extracts at 500 mg/kg significantly increased survival of tumor-bearing mice, however a clear survival advantage in the group treated with the butanolic extract was observed. These results suggest that A. australe may exert effects on myelopoiesis that may be implicated in antitumor immune responses.     

Evaluation of Neolamarckia cadamba (Roxb.) Bosser leaf extract on glucose tolerance in glucose-induced hyperglycemic mice

Neolamarckia cadamba (Rubiaceae) leaf is used in folk medicine of Bangladesh for the treatment of diabetes, but so far no scientific study has been done which may support its use in traditional medicine. The present study was carried out to evaluate the possible glucose tolerance efficacy of methanolic extract of Neolamarckia cadamba leaf using glucose-induced hyperglycemic mice. The extract at different doses was administered one hour prior to glucose administration and blood glucose level was measured after two hours of glucose administration (p.o.) using glucose oxidase method. The statistical data indicated significant oral hypoglycemic activity on glucose-loaded mice at the two highest doses of 200 and 400 mg extract per kg body weight. Maximum anti-hyperglycemic activity was shown at 400 mg per kg body weight, which was comparable to that of, glibenclamide (10 mg/kg). The methanolic extract of leaf of Neolamarckia cadamba had beneficial effects in reducing the elevated blood glucose level of hyperglycemic mice.     

Apoptotic effect of organophosphorus insecticide chlorpyrifos on mouse retina in vivo via oxidative stress and protection of combination of vitamins C and E

Organophosphorus insecticide poisoning is widely investigated, and a growing number of evidence indicates its effects to cause ocular lesions, but the mechanisms of its ocular effects are not well elucidated. Here, effects of organophosphorus insecticide chlorpyrifos on mouse retina in vivo and protection of combination of vitamins C and E were reported. Cell apoptosis, lipid peroxidation and DNA damage were increased, and activities of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase were decreased in retina of chlorpyrifos-administrated mice (63 mg/kg, single treatment, via oral gavage). Pretreatment of combination of antioxidants vitamin C (250 mg/kg) and vitamin E (150 mg/kg) (once daily for 6 days, hypodermic injecting) significantly attenuated these effects of chlorpyrifos, demonstrating oxidative stress was involved in chlorpyrifos-induced cell apoptosis in mouse retina. Moreover, chlorpyrifos treatment inhibited acetylcholinesterase activity and promoted [Ca²⁺]_i level in mouse retinal cells, which were also attenuated by combination of vitamins C and E. These results may have implications for treatment of organophosphorus insecticide poisoning in retina with combination of vitamins C and E.     

Effect of Delonix regia leaf extract on glucose tolerance in glucose-induced hyperglycemic mice

Delonix regia (Fabaceae) leaf is used in folk medicine of Bangladesh for the treatment of diabetes, but so far no scientific study has been done which may support its use in traditional medicine. The present study was carried out to evaluate the possible glucose tolerance efficacy of methanolic extract of Delonix regia leaf using glucose-induced hyperglycemic mice. The extract at different doses was administered one hr prior to glucose administration and blood glucose level was measured after two hrs of glucose administration (p.o.) using glucose oxidase method. The statistical data indicated significant oral hypoglycemic activity on glucose-loaded mice at every dose. Maximum anti-hyperglycemic activity was showed at 400 mg/kg which was comparable to that of a standard drug, glibenclamide (10 mg/kg). The methanolic extract of leaf of Delonix regia had beneficial effects in reducing the elevated blood glucose level of hyperglycemic mice.     

Antiplasmodial activity of Xanthium strumarium against Plasmodium berghei-infected BALB/c mice

The present work was undertaken to evaluate the antiplasmodial activity of ethanolic leaves extract of traditional medicinal plant Xanthium strumarium in Plasmodium berghei-infected BALB/c mice along with phytochemical screening and acute toxicity test to support its traditional medicinal use as a malaria remedy. The ethanolic leaves extract of X. strumarium (ELEXS) 150, 250, 350 and 500 mg/kg/day demonstrated dose-dependent chemosuppression during early and established infection long with significant (p < 0.001) repository activity. The oral administration of 500 mg/kg/day concentration showed a maximum of 88.6% chemosuppression during early infection, which was more than that of the standard drug chloroquine (5 mg/kg/day) with 88.3% chemosuppression. However, 60% mortality has been found in this group. The LD₅₀ of ELEXS was found to be 1.5 g/kg/mouse. The administration of 350 mg/kg/day concentration of extract have been found to exert 90.40% chemosuppression during repository infection, which was well comparable to standard drug pyrimethamine (1.2 mg/kg/day) exerting 92.91% chemosuppression. The extract has been found to enhance mean survival time of mice from 21 to 26 days with 250 and 350 mg/kg/day concentrations, while 150 mg/kg/day concentration has been found to sustain all the mice up to 29 days which was similar to the employed standard drug chloroquine (5 mg/kg/day). All these findings support the ethanopharmacological use of X. strumarium as malarial remedy and indicate the potential of plant for active antiplasmodial components.     

Inhibitory effects of roxithromycin on tumor angiogenesis, growth and metastasis of mouse B16 melanoma cells

We examined the effects of roxithromycin, a 14-membered ring macrolide antibiotic, on tumor angiogenesis, tumor growth and metastasis of mouse B16BL6 melanoma cells. The inhibitory effect of roxithromycin on angiogenesis using mouse dorsal air sac model was dose-dependent, and 100 mg/kg of roxithromycin administered intraperitoneally twice a day reduced the dense capillary network area to about 20% of the control. Administration of roxithromycin histologically reduced the development of microvessels and mononuclear cell infiltration. In vivo tumor growth studies demonstrated that intraperitoneal administration of roxithromycin at 20 mg/kg/day and 50 mg/kg/day reduced tumor size of B16BL6 melanoma to about 56% and 33% (experiment 1), 71% and 48% (experiment 2) of that in the respective controls. Roxithromycin also significantly inhibited pulmonary metastasis of B16BL6 cells in a spontaneous system. The inhibitory activities of roxithromycin on angiogenesis, tumor growth and metastasis were compared with those of a potent angiogenesis inhibitor, TNP-470. These data demonstrated that roxithromycin has potent antiangiogenic and antitumor effects and might have possible therapeutic applications.     

IMMUNIZATION OF MICE AGAINST ALMONELLA TYPHIMURIUM USING DIFFERENT DNA PREPARATIONS

Groups of female BALB/c mice were given primary and booster injections of whole genomic DNA extracted from S. typhimurium, P. aeruginosa, or S. aureus. Other groups of mice were immunized in a similar manner with the 1.57kb fragment of the mouse virulence gene (mviA), pTargeT vector (plasmid DNA)/1.57kb construct, pTargeT vector, or saline. Mice in all groups were challenged intraperitoneally with 100 LD50 of S. typhimurium. The bacterial genomic DNA was extracted using the Pure Gene extraction kit. Specific primers were used to amplify the 1.57kb fragment by PCR. The pTargeT Mammalian Expression Vector System was used to prepare the plasmid/1.57kb construct. Bacterial genomic DNA extracted from P. aeruginosa and S. aureus appeared to induce non-specific resistance in mice. Specific, in addition to non-specific resistance appeared to be induced when genomic DNA from S. typhimurium was used. There was a prolongation of survival in the groups of mice that received either the 1.57kb fragment or the pTargeT vector/1.57kb construct and 16.67% and 33.34% respectively, of mice in each group survived at 40 days post challenge. None of the mice in the saline control group survived by day 7 post challenge.

It is suggested that the non-specific resistance observed in this study might have been due to the adjuvant effect of the non-methylated CpG and other immunostimulatory motifs in bacterial DNA. Specific resistance obtained when genomic DNA from S. typhimurium was used might have been due to minute antigenic contamination, or virulence factor genes other than the mviA gene, might have been expressed in the host, which induced specific immunity.     

Suppression of type II collagen-induced arthritis by a new isocoumarin, NM-3

The anti-arthritic effect of NM-3, a new isocoumarin, was examined using a type II collagen-induced arthritis model for human rheumatoid arthritis in DBA/1J mice. NM-3 by oral administration suppressed dose-dependently (2-20 mg/kg/day) not only macroscopic changes such as erythema and swelling of limbs but also histopathologic changes and radiographic changes such as bone lesions. The efficacy of NM-3 was greater than those of disease-modifying anti-rheumatoid drugs (DMARDs), auranofin (40 mg/kg/day) and bucillamine (10 mg/kg/day). NM-3 failed to suppress carageenan-induced edema and to inhibit the activities of inflammation-related enzymes including cyclooxygenase-1 and -2, 5-lipoxygenase and phospholipase A2, suggesting that the mode of anti-arthritic action of NM-3 may be different from those of non-steroidal anti-inflammatory agents (NSAIDs). Since NM-3 inhibits angiogenesis in a mouse dorsal air-sac model, the observed anti-arthritic effect of NM-3 might be partly attributed to the antiangiogenic activity. Thus, NM-3 is a potential orally active therapeutic agent for the treatment of human rheumatoid arthritis.     

Dichloroacetate reverses the hypoxic adaptation to bevacizumab and enhances its antitumor effects in mouse xenografts

Inhibition of vascular endothelial growth factor increases response rates to chemotherapy and progression-free survival in glioblastoma. However, resistance invariably occurs, prompting the urgent need for identification of synergizing agents. One possible strategy is to understand tumor adaptation to microenvironmental changes induced by antiangiogenic drugs and test agents that exploit this process. We used an in vivo glioblastoma-derived xenograft model of tumor escape in presence of continuous treatment with bevacizumab. U87-MG or U118-MG cells were subcutaneously implanted into either BALB/c SCID or athymic nude mice. Bevacizumab was given by intraperitoneal injection every 3 days (2.5 mg/kg/dose) and/or dichloroacetate (DCA) was administered by oral gavage twice daily (50 mg/kg/dose) when tumor volumes reached 0.3 cm³ and continued until tumors reached approximately 1.5–2.0 cm³. Microarray analysis of resistant U87 tumors revealed coordinated changes at the level of metabolic genes, in particular, a widening gap between glycolysis and mitochondrial respiration. There was a highly significant difference between U87-MG-implanted athymic nude mice 1 week after drug treatment. By 2 weeks of treatment, bevacizumab and DCA together dramatically blocked tumor growth compared to either drug alone. Similar results were seen in athymic nude mice implanted with U118-MG cells. We demonstrate for the first time that reversal of the bevacizumab-induced shift in metabolism using DCA is detrimental to neoplastic growth in vivo. As DCA is viewed as a promising agent targeting tumor metabolism, our data establish the timely proof of concept that combining it with antiangiogenic therapy represents a potent antineoplastic strategy.