Abstract  The present study was conducted to evaluate the antioxidant, anti-inflammatory, and antiproliferative activities of organic fractions from Cystoseira sedoides (Desfontaines) C. Agardh . Various fractions of C. sedoides (chloroform (F-CHCl(3)), ethyl acetate (F-AcOEt), and methanol (F-MeOH)) were screened for total phenol content, as well as antioxidant activity, using the stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH), and assays for determining the reducing power of these fractions. The anti-inflammatory properties of these fractions were assessed using the carrageenan-induced rat paw oedema model. The antiproliferative activity of C. sedoides fractions was evaluated on normal Madin-Darby canine kiney (MDCK), and fibroblast cells and on cancer cell lines (A549, MCF7, and HCT15), using the ability of the cells to metabolically reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) formazan dyes. The F-CHCl(3) and F-AcOEt fractions showed significant total phenolic content at 55.09 and 61.30 mg gallic-acid equivalent/g dried sample, respectively. Using the DPPH method, the F-CHCl(3) and the F-AcOEt fractions exhibited the strongest radical scavenging activity, with IC(50) 120 µg/mL for F-CHCl(3) and 121 µg/mL for F-AcOEt, which approaches the activity of the powerful antioxidant standard, Trolox (IC(50) = 90 µg/mL). The reducing power of the samples was in the following order: F-AcOEt > F-CHCl(3 ) > F-MeOH fraction. The F-CHCl(3) and F-AcOEt fractions of C. sedoides tested at different doses (25 and 50 mg/kg, intraperitoneally (i.p)), exhibited a dose-dependent reduction of rat paw oedema. The percentage of inhibition of oedema, 3 h after carrageenan injection, ranged from 67.71% to 73.49% and from 67.74% to 74.58%, for F-CHCl(3) and F-AcOEt, respectively. Their effects are comparable with that of lysine acetylsalicylate (300 mg/kg body mass; i.p.), which is used as a reference drug with the ability to inhibit oedema by 66.14%. Our results revealed that the F-CHCl(3) and F-AcOEt fractions from C. sedoides showed important antiproliferative properties towards all of the cancer cell lines studied here, as judged by their IC(50) values, which ranged from 52.6 to 66.5 µg/mL for A549; 22.4 to 70.2 µg/mL for MCF7, and 250.6 to 255.3 µg/mL for HCT15. Moreover, no visible destruction or alteration of normal cells was observed, even at 500 µg/mL F-CHCl(3) or F-AcOEt. These results suggest that C. sedoides fractions might be used as a potential source of natural antioxidant, anti-inflammatory, and antitumor agents. The purification and determination of the chemical structures of the compounds in these active fractions are under investigation. The results could provide a compound(s) with a promising role in future medicines and nutrition, when used either as a drug or a dietary supplement.

Abstract  This study is designed to examine the chemical composition of the essential oil and antioxidant activities of the different extracts of Tanacetum sonbolii Mozaff. from Iran for the first time. The essential oil was isolated by hydrodistillation and its gas chromatography and gas chromatography-mass spectrometry analyses resulted in the identification of 26 components, representing 96.5% of the oil. The major components were characterised to be α-cadinol (35.3%), globulol (20.1%) and 1,8-cineole (8.6%). Antioxidant activities of the various extracts of the plant were determined by two different test systems; 1,1-diphenyl-2-picrylhydrazyl (DPPH) and β-carotene-linoleic acid. Also, their total phenolic and flavonoid contents were determined. DPPH radical-scavenging activities of test samples followed the order water > chloroform > ethyl acetate > butanol > BHT > methanol. Moreover, the ethyl acetate extract showed better β-carotene bleaching capacity than the other extracts and the amount of total phenolics was very high in ethyl acetate extract.

Abstract  The antimicrobial activities of Circium arvense extracts were evaluated against two human gram positive (Staphylococcus aureus, Micrococcus luteus) and four gram negative pathogen (Escherichia coli, pseudomonas aeruginosa, Enterobacter, Klebsiella pneumoniae). The Methanol extracts of Circium arvense were used to obtain various fractions (X1) n-hexane, (X2) chloroform, (X3) ethyl acetate and (X4) n-butanol. The agar well diffusion assay and agar dilution susceptibility testing were carried out to determine the zone of inhibitions and the minimum inhibitory concentration respectively. Antifungal activity was employed by agar well diffusion method. The Ampicilline, Ofloxacine and Itraconazole were used as standard agents. Almost all fractions exhibited more or less antimicrobial activity. The X2 (Chloroform) fraction was reported as highly active fraction with good antimicrobial activity MIC 0.312 mg/ml against staphylococcus areous. The MBC values of fraction X2 (chloroform) was 2 times the MIC. Similarly all fractions exhibited good inhibitory activities against Aspergillus niger. The fraction X1(n-hexane) was found highly active 6(±0.11), followed by X2 (chloroform) fraction 5 (±0.17). Present study provides a base to explore the antimicrobial potential of Circium arvense. It is concluded that the Circium arvense having potential antibacterial and antifungal activities, irrespective of fact that the MIC values reported were high. Further investigations are recommended to exploit the hidden medicinal value of plant.

Abstract  The self-assembly of core-substituted naphthalene diimides bearing triethylene glycol motifs leads to the formation of stable vesicles in DMSO and CHCl(3)/MeOH (6 : 4, v/v) solvents. The vesicles were evaluated by means of UV/vis and fluorescence spectroscopy, transmission electron microscopy, atomic force microscopy and dynamic light scattering.

Abstract  ETHNOPHARMACOLOGICAL RELEVANCE: To study the potential benefit of the traditional Mexican medicinal plant Galium mexicanum Kunth (Rubiaceae). Hexane, chloroform, and methanol extracts as well as various fractions from these extracts were tested to determine antibacterial, antifungal, antiparasitic or anti-inflammatory activities in vitro.

MATERIALS AND METHODS: Aerial parts of the plant were extracted with various solvents and fractionated accordingly. Their antibacterial and antifungal activities were assessed on nine bacterial and four fungal strains. Leishmania donovani was used as a protozoan strain for antiparasitic activity. The anti-inflammatory activity of the compounds was investigated by measuring the secretion of interleukin-6 when macrophages were exposed to lipopolysaccharide.

RESULTS: Various extracts and fractions obtained from this plant exhibit antibacterial, antifungal, antiparasitic, and anti-inflammatory activities. Of special interest was the hexane fraction HE 14 b, which show antibacterial (ranging between 67 and 666 μg/ml) and antifungal (at concentrations of 333 μg/ml) activities. Also the hexane fraction HE 5 exhibited antiparasitic activity (at concentrations of 260 μg/ml), whereas the methanol fraction ME 13-15 showed a potent anti-inflammatory activity when compared to dexamethasone. Chemical analyses of the chloroform extract show the presence of triterpenes, saponins, flavonoids, sesquiterpene lactones, and glucosides, but no tannins were detected in the assayed extract.

CONCLUSIONS: The benefit of Galium mexicanum as a traditional medicinal plant was confirmed using antibacterial and antifungal assays in vitro. We also report for the first time, and to the best of our knowledge, antiparasitic and anti-inflammatory activities of this plant.

Abstract  In 1982, Levin et al. published a paper describing a new Salmonella typhimurium strain, TA102, for detecting mutagenic agents that react preferentially with AT base pairs. This strain has an AT base pair at the critical mutation site within the hisG gene, which is located on a multicopy plasmid, pAQ1; the chromosomal copy of the hisG gene has been deleted. It also has an intact excision repair system, thus facilitatin the detection of cross-linking agents, and carries the mutator plasmid, pKM101. Although TA102 has been shown to be reverted by certain mutagenic agents that are not detected in the usual battery of strains (TA1535, Ta1537, TA1538, TA98 and TA100), there has been a general reluctance within the field to include TA102 as one of the standard screening strains. This may in part result from the difficulties which have been experienced in many laboratories in maintaining the strain, and in obtaining reproducible spontaneous and induced revertant counts. At Glaxo we routinely include certain Escherichia coli strains in our mirobial test battery, and were aware that some of the genetic features offered by TA 102 were already being covered by these strains. For examples, E. coli WP2 (pKM101) has an AT base pair at the critical mutation site within the trpE gene, is excision proficient (and thus will detect cross-linking agents) and carries the pKM101 plasmid to enhance error-prone repair. From the published literature it was apparent that a number of the ‘TA102 specific’ mutagens could be detected in E. coli e.g. neocarzinostatin, UV and 8-MOP plus UV. We have expanded this data base by comparing the activity of nine ‘TA102 specific’ mutagens (cumene hydroperoxide, hydrogen peroxide, phenylhydrazine, glutaraldehyde, formaldehyde, Danthron, bleomycin, streptonigrin and mitomycin C) in plate incorporation assays using S.typhimurium TA102, and E. coli WP2 (pKM101) and E. coli WP2 uvrA (pKM101). The results obtained indicate that these E. coli strains, used in combination, will detect a similar spectrum of mutagens as TA102.

Abstract  Numerous epidemiological studies have associated episodes of increased air pollution with increased incidence of respiratory disease, including pneumonia, croup, and bronchitis. Trichloroethylene (TCE) and chloroform are among 33 hazardous air pollutants identified by the U.S. Environmental Protection Agency as presenting the greatest threat to public health in the largest number of urban areas. Also, both are common indoor air pollutants. Here, we assessed the potential effects of TCE and chloroform on resistance to pulmonary bacterial infection and related alveolar macrophage (AM) function. CD-1 mice were exposed by inhalation to filtered air (control) or concentrations of TCE ranging from 5 to 200 ppm, or concentrations of chloroform ranging from 100 to 2000 ppm. Immediately following exposure, mice were challenged with an aerosol of Streptococcus zooepidemicus and monitored for clearance of bacteria from the lung and mortality. In separate experiments, exposed mice were injected intratracheally with viable bacteria and phagocytic function was evaluated in macrophages obtained from lung washes 30 min later. The NOEL for enhanced mortality to infection was 25 ppm for TCE and 500 ppm for chloroform. Relative to the air controls, differences in clearance of bacteria from the lung were noted in mice exposed to TCE (NOEL = 50 ppm) and to chloroform (NOEL 100 ppm), and differences in AM phagocytic index were noted for TCE (NOEL = 100 ppm) and for chloroform (NOEL < 100 ppm). The data support the utility of the S. zooepidemicus infectivity model in assessing potential increased risk of respiratory infection and suggest that delayed clearance of bacteria from the lung or decreased phagocytosis are viable alternatives to mortality as an endpoint. Collectively, these endpoints are among the most sensitive health effects reported for TCE.

Abstract  Centaurea L. (Astreaceae) species are used as herbal remedies in Turkey. Centaurea calolepis Boiss. is an endemic species of Anatolia that has not been subjected to phytochemical studies except essential oil analysis.

Secondary metabolite determination, isolation and structure elucidation of pure compounds were performed on C. calolepis. Cnicin, which is the main component of several Centaurea species, was tested for its in vitro anti-inflammatory, antioxidant and cytotoxic activities.

Chloroform and methanol extracts of the aerial parts of C. calolepis were subjected to isolation process using column chromatography. The structures of the compounds were characterized by 1D- and 2D-NMR experiments. Thin-layer chromatography and high performance liquid chromatography were used in determination of phenolics. Cnicin was subjected to a panel of cellular assays to test for inhibition of nuclear factor κB (NF-κB), inducible nitric oxide synthase (iNOS), reactive oxygen species and cytotoxicity.

Cnicin, lucenin-2, schaftoside and 3-O-feruloylquinic acid were isolated from C. calolepis extracts. Vicenin-2, vitexin, isovitexin, homoorientin, rutin, orientin, luteolin-7-O-glycoside and chlorogenic acid were determined in fractions. Cnicin showed inhibition of NF-κB and inhibition of iNOS activity with IC₅₀ Values of 1.8 and 6.5 µM, respectively. Cytotoxic activity of cnicin was observed toward pig kidney epithelial (LLC-PK₁₁), human malignant melanoma (SK-MEL) and human ductal carcinoma (BT-549) cells with IC₅₀ values of 23.3, 14.0 and 18.3 µM, respectively.

This is the first detailed report of secondary metabolites of C. calolepis. Evaluation of biological activity of cnicin establishes the potential of this compound as an anti-inflammatory and cytotoxic agent.

Abstract  The antispasmodic effects of hexane-, chloroform-, methanol- and water-extracts of the orchid Encyclia michuacana were studied in vitro on guinea pig ileum against three spasmogens: acetylcholine (Ach), histamine and barium chloride. The chloroform extract exerted a significant antispasmodic effect on ileum contractions induced by Ach, histamine and barium chloride (IC(50) = 90.64, 73.12 and 115.2 microg/mL, respectively). Furthermore, the chloroform extract of E. michuacana provoked a concentration-dependent inhibition of spontaneous contractions of guinea pig ileum with potencies comparable to those of papaverine. The antagonism against the spasmogens used suggests that the action of the chloroform extract of E. michuacana could be due mainly to the presence of gigantol. Hexane-, methanol- and water-extracts did not elicit any significant spasmolytic activity.

Abstract  The frequency and mutational profile of H-ras gene activation were determined in spontaneous liver tumors of male C57BL/6 x C3H/He mice and in tumors induced with the genotoxic hepatocarcinogen benzidine.2 HCl or the nongenotoxic hepatocarcinogens phenobarbital, chloroform, and ciprofibrate. DNA sequence analysis of the H-ras gene from representative tumors revealed that 32 of 50 (64%) spontaneous tumors and 13 of 22 (59%) benzidine.2 HCl-induced tumors contained a point mutation in codon 61. Tumors induced with the nongenotoxic agents had a much lower frequency of codon 61 mutations, i.e., phenobarbital, 1 of 15 (7%); chloroform, 5 of 24 (21%), and ciprofibrate, 8 of 39 (21%). No mutations were observed at codons 12, 13, and 117 in tumors from any of the groups. Only three base pair substitutions within codon 61 were found. The one most frequently detected in all of the groups was a C.G to A.T transversion at the first nucleotide position, occurring at a 59%, 85%, 100%, 80%, and 88% frequency in the spontaneous tumors and in the tumors induced with benzidine 2.Hcl, phenobarbital, chloroform, and ciprofibrate, respectively. In these same groups an A.T to G.C transition or an A.T to T.A transversion at the second nucleotide position occurred at a frequency of 34%, 8%, 0%, 0%, and 12%, and 6%, 8%, 0%, 20%, and 0%, respectively. The number of tumors carrying an activated H-ras gene in the nongenotoxic treatment groups is within the range that would be expected if those animals had not received any treatment. This indicates that the activation of the H-ras gene in those tumors is probably the result of a spontaneous event. The data suggest that these toxicologically and pharmacologically diverse nongenotoxic hepatocarcinogens increase the frequency of liver tumors but do not induce mutations in the H-ras gene. Instead these agents appear to interact with a population of cells that do not contain an activated H-ras gene. This suggests that the mechanisms of tumor development by these nongenotoxic carcinogens differ at least partially from the mechanisms responsible for the development of spontaneous tumors or those induced by a typical genotoxic agent.

Abstract  Simaba ferruginea A. St-Hil. (Simaroubaceae) is a subshrub typical of the Brazilian Cerrado, whose rhizomes are popularly used as infusion or decoction for the treatment of gastric ulcers, diarrhea and fever.

To evaluate the pharmacological mechanism(s) of action of the antiulcer effects of the methanol extract of Simaba ferruginea and its alkaloid canthin-6-one.

Rhizome of Simaba ferruginea was macerated with methanol to obtain the methanol extract (MESf) from which was obtained, the chloroform fraction. Canthin-6-one alkaloid (Cant) was purified and then isolated from the chloroform fraction (CFSf). The isolated Cant was identified by HPLC. Anti-ulcer assays were determined using ethanol and indomethacin-induced ulcer models in mice and rats respectively. In order to determine the probable mechanisms of actions of MESf and Cant animals were pretreated with l-NAME prior to anti-ulcer agent treatments and ulcer induction and nitric oxide (NO) level determined in order to assess NO involvement in the gastroprotective effects. Assays of malondialdehyde (MDA), myeloperoxidase (MPO), pro-inflammatory cytokines: interleukin 8 (IL-8) and tumor necrosis factor-alpha (TNF-α) and prostaglandin E(2) (PGE(2)) were also carried out according to previously described methods.

The results indicate that the antiulcerogenic effects of MESf and Cant in ethanol-induced ulcer is mediated in part through increase in the production of protective endogenous NO as the antiulcerogenic activity of MESf and Cant was reduced in animals pre-treated with l-NAME. In indomethacin-induced ulcer pre-treatment with MESf and Cant showed reduction in the levels of MPO and MDA in the gastric tissue, thus indicating the participation of the antioxidant mechanisms on the gastroprotective effects. The plasma levels of IL-8 in ulcerated rats with indomethacin were also reduced by Cant, but not by MESf, indicating that inhibition of this cytokine contributes to the gastroprotective effect of Cant. However MESf and Cant had no effect on the mucosal membrane levels of PGE(2), indicating that the gastroprotective effects of these agents is independent of PGE(2) modulation.

The results obtained in this study with MESf and Cant added insights into the pharmacological mechanisms involved in their mode of antiulcer action. The results indicate that Cant is one of the compounds responsible for these effects. Such findings are of extreme importance in the strive for future development of potent, safer and effective antiulcer agent. The efficacy of MESf and Cant in gastroprotection shows that Simaba ferruginea might be a promising antiulcer herbal medicine, in addition to confirming the popular use of this plant against gastric ulcer models utilised in this study.

Abstract  Partition coefficients are required for developing physiologically based pharmacokinetic models used to assess the uptake, distribution, tabolism, and elimination of volatile chemicals in mammals. A gas-phase vial equilibration technique is presented for determining the liquid:air and tissue:air partition coefficients for low-molecular-weight volatile chemicals. This technique was developed from two previously described medium:air methods, relied solely on measurement of chemical concentration in the gas phase, and, compared to earlier work, extends the range of chemicals and tissues examined. Partition coefficients were determined with 0.9% saline, olive oil, and blood, liver, muscle, and fat tissues from rats for 55 compounds. Human blood:air coefficients were determined for 36 compounds and several blood:air values were also determined in the mouse and for one compound in the hamster. An approach is described for predicting the tissue solubilities of untested compounds based on oil:air and saline:air coefficients using regression analyses. A similar approach is used to model fat:air coefficients in terms of oil:air values and to model human blood: air coefficients in terms of rat blood:air coefficients.

Abstract  The results of a preliminary rangefinding 13-wk oral toxicity study and of two longer term studies on chloroform in toothpaste base are reported. Significant changes in serum enzymes and certain haemotological parameters were seen at the higher dose-levels in the rangefinding study. Intercurrent disease made it necessary to terminate the first long-term experiment prematurely after 1 yr. No evidence of serious toxicity was recorded. In the second long-term experiment, groups of 50 caesarian-derived SPF Sprague-Dawley rats of each sex received either the equivalent of 60 mg CHCl3/kg/d in toothpaste base or the vehicle only, by gavage on 6 d/wk for 80 wk and were then observed for up to a further 15 wk. Chloroform-treated rats of both sexes survived better than the controls, though both groups had a high incidence of non-neoplastic respiratory and renal disease. Female rats gave a consistent finding of decrease in plasma cholinesterase, shown to be related to activity against butyrylcholine but not acetyl-beta-methylcholine. Tumours of various sites were seen in 39 percent of chloroform-treated rats of both sexes examined histologically, compared with 38 percent of vehicle controls. There were no treatment-related effects on the incidence of liver or kidney tumours. Histologically-malignant mammary tumours were reported in more treated than control rats, but the difference in incidence was not statistically significant.

Abstract  Two strains of Escherichia coli, WP2p and WP2uvrA−p, were treated with chloroform in the plate incorporation assay, and in liquid pre-incubation tests, and in both cases the chemical did not induce reversions. Chloroform, with metabolic activation (addition of S-9 mix), also failed to induce chromosome breakage or sister-chromatid exchanges in human lymphocytes, but benzo[a]pyrene, with and without S-9 mix, induced significant chromosome breakage and sister-chromatid exchanges in lymphocytes from the same donor. The relevance of these results in the light of other negative in vitro tests on chloroform, and positive and negative carcinogenicity tests, is discussed.

Abstract  Chloroform was tested for mutagenicity in the Salmonella/microsome assay using five strains of Salmonella typhimurium. In view of previous reports describing the development of liver and kidney tumours in some experiments involving long-term administration of chloroform to rats and mice, the mutagenicity tests were carried out in the absence of any S-9 microsomal-enzyme preparation and in the presence of S-9 microsomal-enzyme preparations derived from (a) livers and (b) kidneys of rats and mice previously exposed to the microsomal-enzyme inducer Aroclor 1254. No evidence of potential mutagenicity was observed under any of the test conditions. To determine whether the findings might have been influenced by the volatility of the chloroform, the test organisms were exposed to chloroform vapour, but again chloroform gave no indication of potential mutagenicity. Taken in conjunction with already published data from mutagenicity studies with chloroform, it appears unlikely that the tumours observed in some long-term rodent studies are attributable to a genotoxic action of the compound.

Abstract  There is sufficient evidence that anesthetics may cause cancer to justify a test of their carcinogenic potential. Baden, et al., using the Ames test, a rapid and inexpensive genetic indicator of carcinogenicity, have shown that among currently used anesthetics fluroxene alone caused bacterial mutations. The authors used the sister chromatid exchange (SCE) technique, another rapid assay of mutagenic-carcinogenic potential. The frequency of sister chromatid exchanges in Chinese hamster ovary cells increases when the cell cultures are exposed to mutagen-carcinogens, particularly in the presence of a metabolic activating system. With this test system a one-hour exposure to 1 MAC nitrous oxide, diethyl ether, trichloroethylene, halothane, enflurane, isoflurane, methoxyflurane, or chloroform did not increase SCE values. Divinyl ether, fluroxene and ethyl vinyl ether increased SCE values in the same circumstances. Results of this study of mammalian cells suggest that no currently used anesthetic is a mutagen-carcinogen. The results also suggest that anesthetics containing a vinyl moiety may be mutagen-carcinogens.

Abstract  To evaluate the applicability of an in vivo-in vitro replicative DNA synthesis (RDS) test using rat hepatocytes, we conducted the RDS test with 22 nongenotoxic (Ames-negative) hepatocarcinogens and 25 noncarcinogens under our standardized conditions and judgement criteria. Compared to controls (RDS incidence of under 1.0%), the RDS test gave positive results for 18 hepatocarcinogens (positive sensitivity: 82%), and negative results for 20 noncarcinogens (negative specificity: 80%), and thus the overall concordance was 81%. These findings strongly suggest that the RDS test is an extremely useful method for early detection of nongenotoxic hepatocarcinogens.

Abstract  Chloroform, also known as trichloromethane, is primarily used (93%) in the manufacture of fluorocarbons for refrigerants, propellants, and plastics. The remainder is used for many purposes including extracting and purifying antibiotics, as an industrial solvent, in preparation of dyes, drugs and pesticides, as a component of some toothpastes, cough medicines, liniments, salves, in photographic processing and in industrial drycleaning. A carcinogenesis bioassay of USP grade chloroform was conducted using Osborne-Mendel rats and B6C3F1 mice. Chloroform was administered orally (by gavage) in corn oil to 50 animals of each sex and at two dose levels five times per week for 78 weeks. Rats were started on test at 52 days of age and sacrificed after 111 weeks. The dose levels for males were 90 and 180 mg/kg body weight. Female rats were started at 125 and 250 mg/kg, reduced to 90 and 180 mg/kg after 22 weeks, with an average level of 100 and 200 mg/kg for the study. A decrease in survival rate and weight gain was evident for all treated groups. The most significant observation (P=.0016) was kidney epithelial tumors in male rats with incidences of: 0% in controls, 8% in the low dose and 24% in the high dose groups. Although an increase in thyroid tumors was also observed in treated female rats, this finding was not considered biologically significant. Mice were started on test at 35 days and sacrificed after 92-93 weeks. Initial dose levels were 100 and 200 mg/kg for males and 200 and 400 mg/kg or female mice. These levels were increased after 18 weeks to 150/300 and 250/500 mg/kg respectively so that the average levels were 138 and 277 mg/kg for males and 238 and 477 mg/kg for female mice. Survival rates and weight gains were comparable for all groups except high dose females which had a decreased survival. Highly significant increases (P<.001) in hepatocellular carcinoma were observed in both sexes of mice with incidences of: 98% and 95% for males and females at the high dose; 36% and 80% for males and females at the low dose as compared with 6% in both matched and colony control males, 0% in matched control females and 1% in colony control females. Nodular hyperplasia of the liver was observed in many low dose male mice that had not developed hepatocellular carcinoma. Levels of Evidence of Carcinogenicity: MaleRats: Positive FemaleRats: Negative MaleMice: Positive FemaleMice: Positive

Abstract  The volatile solvents carbon tetrachloride and chloroform are carcinogens that are often reported as nonmutagenic in bacterial mutagenicity assays. In this study, we evaluated the mutagenicity of these compounds in Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA/pKM101 and WP2/pKM101, with and without S9 mix, using a gas exposure method. Tests were also conducted with a glutathione-supplemented S9 mix. Carbon tetrachloride was mutagenic in TA98 without S9 mix, and in WP2/ pKM101 and WP2uvrA/pKM101 with and without S9 mix; carbon tetrachloride was not mutagenic in TA100, TA1535 or TA1537. Chloroform was mutagenic in WP2/pKM101, but only in the presence of glutathione-supplemented S9 mix. Chloroform was not mutagenic in TA98, TA100, TA1535, TA1537, or WP2uvrA/pKM101 with or without S9 mix, and was not mutagenic in TA98, TA100, TA1535, TA1537, or WP2uvrA/pKM101 in the presence of glutathione-supplemented S9 mix. The data indicate that carbon tetrachloride and chloroform are bacterial mutagens when adequate exposure conditions are employed and suggest that a genotoxic mode of action could contribute to the carcinogenicity of these compounds.

Abstract  BACKGROUND: Callistemon citrinus Curtis belongs to family Myrtaceae that has a great medicinal importance. In our previous work, fruits of Callistemon citrinus were reported to have relaxant (antispasmodic) activity. The current work describes the screening of fractions of the crude methanol extract for tracing spasmolytic constituents so that it shall help us for isolation of bioactive compounds. Acute toxicity and brine shrimp cytotoxicity of crude methanol extract are also performed to standardize it.

METHODS: The crude methanol extract was obtained by maceration with distilled water (500 ml) three times and fractionated successively with n-hexane, chloroform, ethyl acetate and n-butanol (300 ml of each solvent). Phytochemical analysis for crude methanol extract was performed. Acute toxicity studies were performed in mice. Brine shrimp cytotoxicity studies were performed to determine its cytotoxicity and standardize it. In other series of experiments, rabbits' jejunum preparations were used in screening for possible relaxant activities of various fractions. They were applied in concentrations of 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 5.0 and 10.0 mg/ml on spontaneous rabbits' jejunum preparations. In similar fashion, fractions were also tested on KCl (80 mM) -induced contractions. Calcium chloride curves were constructed in K-rich Tyrode's solution. The effects of various fractions were tested on calcium chloride curves at concentrations 1.0, 3.0, 5.0 and 10.0 mg/ml. Curves of verapamil used as reference drug at concentration 0.1 μM and 0.3 μM were also constructed. The curves were compared with their respective controls for possible right shift.

RESULTS: Methanol extract tested strongly positive for saponins and tannins. However, it tested mild positive for presence of proteins, amino acids, carbohydrates and phenolic compounds. LD(50) value for crude methanol extract is 476.25 ± 10.3 (470-481, n = 4) mg/ml. Similarly, EC(50) value for brine shrimp cytotoxicity is 65.5 ± 7.28 (60.8- 69.4, n = 4) mg/ml. All the fractions relaxed the spontaneous and KCl-induced contractions. EC(50) values (mg/ml) for effects of ethyl acetate fraction on spontaneous and KCl induced contractions are 2.62 ± 0.78 (2.15-3.0, n = 4) and 3.72 ± 0.86 (3.38-4.28, n = 4) respectively. Respective EC(50) values (mg/ml) for n-butanol fraction are 3.59 ± 0.2(3.07-3.9, n = 4) for spontaneous, and 5.57 ± 0.2 (5.07-6.11, n = 4) for KCl- induced contractions. EC(50) value for control calcium chloride curve (without extract) is -2.73 ± 0.19 (-2.6 - -2.81, n = 4) while EC(50) for curves treated with 5.0 mg/ml of chloroform is -2.22 ± 0.02 (-2.16 - -2.3, n = 4). EC(50) value for ethyl acetate treated (1.0 mg/ml) tissues is -1.95 ± 0.10 (-1.88 - -2.0, n = 4) vs. control EC(50) = -2.71 ± 0.08 (-2.66 - -2.76, n = 4). All the fractions, except n-hexane, showed a right shift like that of verapamil (EC(50) = -1.72 ± 0.15 (-1.62 - -1.8, n = 4) vs. Control EC(50) = -2.41 ± 0.06 (-2.38 - - 2.44, n = 4), a standard drug that blocks voltage operated calcium channels.

CONCLUSION: Relaxant constituents were more concentrated in ethylacetate fraction followed by chloroform, n -butanol and aqueous fractions that warrant for its isolation. The crude methanol extract is safe at concentration 250 mg/ml or below and results of brine shrimp cytotoxicity assay imply the plant specie may be a source of cytotoxic agents.

Abstract  Methanolic extract of Onosma griffithii and its fractions were evaluated for possible effects on rabbits' jejunum preparations. Rabbits of either sex (weight 1.5-2.0 kg) were used in experiments. Studies were carried out on rabbits' jejunum preparations. Crude methanolic extract of Onosma griffithii (Meth.OG) was tried in concentrations of 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 5.0 and 10.0 mg/ml on rabbits' jejunum preparations. Meth.OG was also tried on KCl-induced contractions to explain its possible mode of actions in the presence and absence of atropine (0.03 µM). Fractions of Meth.OG were tried in similar manner. Calcium chloride curves were constructed for Meth.OG treated tissues that were compared with curves constructed for verapamil in same fashion. Preliminary phytochemical screening of the plant was also performed. Meth.OG increased the amplitude of spontaneous activity of rabbits' jejunum preparations at concentrations of 0.1, 0.3 and 1.0 mg/ml. However, spasmolytic effects were observed at higher concentrations 3.0, 5.0 and 10.0 mg/ml. Mean EC(50) values (mg/ml), respectively, in absence and presence of atropine were 7.5 ± 0.25 (6.9-8.4, n=6) and 3.0 ± 0.17 (2.3-3.5, n=6, p<0.05). Mean EC(50) values, respectively, for effects on spontaneous and KCl-induced contractions were 7.5 ± 0.25 (6.9-8.4, n=6) and 7.3 ± 0.35 (6.25-8.2, n=6, p<0.05). n-Hexane, chloroform and ethyl acetate fractions showed their respective EC(50) values (mg/ml) 9.7 ± 0.25 (8.6-10.2, n=6), 4.0 ± 0.2 (3.5-4.6, n=6) and 1.07 ± 0.093 (0.78-1.5, n=6). EC(50) values for calcium chloride curves in presence of 0.3 mg/ml Meth.OG were - 2.27 ± 0.038 (- 2.4 to - 2.10, n=6) vs. control - 2.78 ±0.04 (-2.9 to - 2.6, n=6, p<0.05) Log [Ca(++)]M. Comparing with curves of calcium chloride constructed in presence of 0.1 µM verapamil, the EC(50) (log [Ca(++)] M) values were - 1.82 ± 0.087 (- 2.0 to - 1.65, n=6) vs. control - 2.64 ± 0.089 (- 2.9 to - 2.4, n=6) demonstrated a right shift (p<0.05). Meth.OG tested positive for terpenes, saponins, sterols, flavonoids and carbohydrates. We concluded that the relaxant effect of Meth.OG is exerted through blocking of calcium channels. However, n-butanolic and aqueous fractions produced spasmogenic effects that require further work for isolation of pharmacologically active substances.

Abstract  OBJECTIVES: The aim of this study was to investigate the flavonoid composition of Scutellaria immaculata and S. ramosissima (Lamiaceae) and the in-vitro biological activity of their extracts and flavonoids.

METHODS: The flavonoid composition of S. immaculata (Si) and S. ramosissima (Sr) were analysed using LC-MS. Antimicrobial activity was studied in vitro against a range of bacteria and fungi using diffusion and microdilution methods. Anti-trypanosomal and cell proliferation inhibitory activity of the extracts and flavonoids was assessed using MTT. The antioxidant activity of the flavonoids and extracts were evaluated using DPPH* test.

KEY FINDINGS: LC-MS investigation of Si and Sr plants allowed the identification, for the first time, of an additional 9 and 16 flavonoids, respectively. The methanol, chloroform and water extracts from these plants and six flavonoids (scutellarin, chrysin, apigenin, apigenin-7-O-glucoside, cynaroside and pinocembrine) exhibited significant inhibition of cell growth against HeLa, HepG-2 and MCF-7 cells. The chloroform extract of Sr showed potent cytotoxic effects with IC50 (drug concentration which resulted in a 50% reduction in cell viability) values of 9.25 ± 1.07 µg/ml, 12.83 ± 1.49 µg/ml and 17.29 ± 1.27 µg/ml, respectively. The highest anti-trypanosomal effect against T. b. brucei was shown by the chloroform extract of Sr with an IC50 (drug concentration which resulted in a 50% inhibition of the biological activity) of 61 µg/ml. The pure flavonoids showed an IC50 range between 3 and 29 µm, with cynaroside as the most active compound with an IC50 value of 3.961 ± 0.133 µm. The chloroform extract of Sr has potent antimicrobial activity against Streptococcus pyogenes (minimum inhibitory concentration, MIC = 0.03 mg/ml). Pinocembrine exhibited a strong activity against the all bacteria except Escherichia coli and yeasts. Water extracts of Sr and Si exhibited potent antioxidant activity with IC50 values of 5.62 ± 0.51 µg/ml and 3.48 ± 0.02 µg/ml, respectively. Scutellarin exerted stronger antioxidant activity than other flavonoids.

CONCLUSIONS: This is the first study reporting an in-vitro biological investigation for Si and Sr. Especially the chloroform extract of Sr showed potent anticancer and antimicrobial activity. Cynaroside had a highly selective and strong cytotoxicity against T. b. brucei while showing only mild effects against cancer cells.

Abstract  In order to improve its stability, immobilized Concanavalin A (Con A) on Toyopearl adsorbents was conjugated with monomethoxy poly(ethylene glycol) succinimidyl propionate (mPEG-SPA) with different molecular weight. A colorimetric method using ninhydrin is proposed to determine the degree of PEGylation; this method has proved to be easy applicable and reproducible. The PEGylation reaction was studied in detail to elucidate how parameters such as molar ratio of mPEG-SPA to Con A and molecular weight of mPEG-SPA affect the degree of PEGylation. The adsorption isotherms of glucose oxidase (GOD) onto native and PEGylated Con A adsorbents showed that the modification did not alter substantially the specificity of the carbohydrate binding ability of Con A. However, the binding capacity for GOD was slightly reduced probably due to the steric hindrance caused by mPEG chains. Adsorption kinetic studies revealed a lower adsorption rate after PEGylation which was attributed to the steric effect. The dynamic adsorption capacity for modified Con A depended very much on the degree of PEGylation and the molecular weight of mPEG derivatives. The adsorption capacity could be highly preserved for Toyopearl Con A modified by mPEG2k (90% of the original adsorption capacity) even with a degree of PEGylation up to 20% (the ratio of primary amino groups of PEGylated immobilized Con A to that of native immobilized Con A). Studies show that the binding capacity of PEGylated Con A was highly preserved under mild process conditions. PEGylated Con A also exhibited obviously higher stability against more stressful conditions such as the exposure to organic solvents and high temperatures. Conjugation of Con A with mPEG2k provided better adsorption performance thus has greater potential for application in affinity separation processes compared with mPEG5k. The fact that PEGylation stabilizes the properties of Con A may greatly expand the range of applications of unstable proteins to bioprocessing (e.g. biocatalysis and downstream separation) as well as other protein applications (e.g. medication, industrial use, etc.).

Abstract  CONTEXT: Artemisia annua L. (Asteraceae), commonly known as sweet wormwood or Qinghao, is an annual herb/shrub native of Asia. The plant grows broadly in Caspian Sea shores in North of Iran. In China, the aerial parts of this plant are source of artemisinin, which is an antimalarial compound.

OBJECTIVE: This study aimed to establish the scientific basis of reported ethnomedicinal use of A. annua as sedative agent.

MATERIAL AND METHODS: The plants were gathered from Gilan Province in Iran. Plant aerial parts were extracted with methanol and concentrated in vacuum. Methanol extract was partitioned into chloroform, petroleum ether, and ethyl acetate. Each fraction was administered intraperitoneally (i.p.) in male mice with different concentrations (50, 100, and 200 mg/kg), and for evaluation of sedative activity, immobility time was determined. In effort to clarify the mechanism of action, flumazenil (3 mg/kg, i.p.) as a benzodiazepine (BZD) receptor antagonist was injected 15 min before chloroform fraction (200 mg/kg, i.p.).

RESULTS: Compared with control group (saline-treated mice), the chloroform fraction significantly increased immobility time in a dose-dependent manner. Flumazenil decreased immobility time induced by chloroform fraction significantly.

DISCUSSION AND CONCLUSION: The results of the present study suggest that A. annua growing in Iran has sedative effects, which are probably mediated via BZD receptors pathways.

Abstract  ETHNOPHARMACOLOGICAL RELEVANCE: In a search for new plant-derived biologically active compounds against protozoan parasites, an ethnopharmacological study was carried out to evaluate extracts from selected 17 traditional medicinal plants which were used by healers from the Garhwal region of North West Himalaya for the treatment of protozoal infections and fever including malaria.

MATERIALS AND METHODS: In vitro activity against erythrocytic stages of Plasmodium falciparum was determined using a modified [3H]-hypoxanthine incorporation assay with the chloroquine- and pyrimethamine-resistant K1 strain. Activity against Trypanosoma brucei rhodesiense was performed on the STIB 900 strain and activity against Trypanosoma cruzi on infected rat skeletal myoblasts (L6 cells) seeded in 96-well microtitre plates while amastigotes of Leishmania donovani strain MHOM/ET/67/L82 were used to assess activity against Leishmania donovani. Cytotoxicity assays were performed against rat skeletal myoblasts (L6-cells).

RESULTS AND CONCLUSIONS: Extracts of Artemisia roxburghiana, Roylea cinerea, Leucas cephalotes, Nepeta hindostana and Viola canescens showed good antiplasmodial activity (IC50<5 μg/ml). The chloroform extract of Artemisia roxburghiana was the most active (IC50 value of 0.42 μg/ml) and the most selective (SI=78) extract for Plasmodium falciparum among all plants extracts examined. The chloroform extract of Leucas cephalotes and the petroleum ether extract of Viola canescens exhibited substantial activities against Leishmania donovani with IC50 values of 3.61 μg/ml (SI=8) and 0.40 μg/ml (SI=30), respectively. The petroleum ether extract of Viola canescens exhibited activity against Trypanosoma cruzi with an IC50 value of 1.86 μg/ml (SI=7). Methanol and water extracts from all plants under investigation were found inactive against all parasites tested. These results support investigation of components of traditional medicines as potential new antiprotozoal agents. On the other hand since herbalism has become the main stream throughout the world, investigation demonstrates that these non-polar plant extracts of six of the plants examined in this study could play an important role in herbal formulations for the treatment of vector borne protozoal diseases.