Breaking the resistance of Escherichia coli: Antimicrobial activity of Berberis lycium Royle.

The antimicrobial activity of root bark of Berberis lycium and its principal component berberine was tested against a panel of microbial strains using agar well diffusion test and further analyzed using micro-broth dilution method. Preliminary analysis, on the basis of zone of Inhibition (ZOI) showed that the methanolic extract of B. lycium was highly effective against Escherichia coli (ZOI 41 ± 1 mm). Among the bacterial strains E. coli was found to be most susceptible and among fungi Candida albicans was the most susceptible for berberine as well as the crude methanolic extract of the plant. Methanolic extract of the plant was more effective for E. coli (MIC 1.7 ± 1.18; MBC 2.4 ± 1.18) than berberine (MIC 3.5 ± 0.57) (p < 0.05), whereas berberine was more effective than crude extracts for C. albicans. In addition, E. coli showed the development of resistant colonies after 72 h when tested with berberine but the development of such colonies was not observed with the methanolic extract of the plant. This could be due to the presence of resistance breaking molecules in the crude methanolic extract of B. lycium. Also the MIC index of crude methanolic extract was 1.39 for E. coli, which showed the mode of action to be bactericidal. HPLC analysis revealed the presence of berberine at highest concentration in methanolic extract of the plant, followed by aqueous extract. Potentiation of this berberine by resistance breaking molecules in the crude extract could be a possible explanation for its strong effectiveness.

Synergistic approach for treatment of chicken coccidiosis using berberine--A plant natural product.

Despite the advent of anticoccidial drugs and vaccines, coccidiosis continues to result in substantial economic losses to the poultry industry. Berberine, a natural alkaloid is well known in studies involving synergistic approaches, thereby reducing the dosage of principal drugs. Therefore, a study was designed to see whether a synergistic anticoccidial effect could be obtained between amprolium and berberine, in vivo using broiler chicken. Anticoccidial activity was measured in comparison to the reference drug amprolium on the basis of oocyst output reduction, mean weight gain and feed conversion ratio. Oocyst output was measured using Mc-Masters counting technique. Different combinations of berberine and amprolium were tested and out of which 1:1 ratio was the most effective for controlling these parasites. Oral gavaging of 100(50 + 50) mg/kg body weight of 1:1 ratio of amprolium and berberine caused the equivalent reduction in number of oocysts (38.85 ± 9.61) one day prior to that of standard drug amprolium (49.95 ± 16.65) as well as pure berberine (44.4 ± 9.61) used in the study. Weight gain of birds was also highest in the synergistic group (1547.43 ± 12.86) among all the infected groups. Besides feed conversion ratio in the synergistic group was also better (1.387 ± 0.026). The results of this study proved the effectiveness of both amprolium and berberine and revealed synergism between amprolium and berberine against coccidian oocysts, confirmed by significant reduction in the number of coccidian oocysts shed in the feces, leading to better weight gain and improved feed conversion ratio. The study deep-rooted the synergistic potential of berberine, a natural bioactive compound for controlling a protozoan parasite and the results of this study corroborate with its use for treatment of severe diarrhoea, amoebiasis and intestinal infections.

Effect of some phytoconstituents on Fe2+/ascorbate induced lipid peroxidation.

Transition metals like iron and copper, present inside the body system play a key role in the production of reactive oxygen radicals. These free radicals, causative agents of lipid peroxidation, not only damage proteins and DNA but also gradually changes the cellular membrane structure and ultimately leads to the loss of function and integrity. Uncontrolled lipid peroxidation results in various age related diseases, malignancy, infective diseases and injuries. Antioxidants and other phytochemical constituents present in the various plants are known to protect cells from such reactive oxygen species (ROS)-mediated damages. Here, we evaluated the effect of certain phytoconstituents present in the well-known medicinal plants on ROS scavenging using rat liver homogenate. The basal lipid peroxidation was found to be 0.1625±0.0095 ngMDA/min/mg protein, which got induced to 0.7938±0.0478 ngMDA/min/mg protein in the presence of Fe2+/ascorbate system. In this context, acteoside, berberine, catechin, 3´5-dihydroxyflavone7-o-ß-D-galacturonide-4-o-ß-D-glucopyranoside (a flavonoid glycoside from cumin), silibin and tetrahydrocurcumin decreased both basal and Fe2+/ascorbate induced lipid peroxidation as determined by thiobarbituric acid reaction. On the other hand, agnuside, andrographolide, picroside-I, negunoside, oleanolic acid, and glycerrihizin, showed enhancement in both basal and induced lipid peroxidation. Phytoconstituents which have decreased both basal and Fe2+/ascorbate induced lipid peroxidation may act as defensive against the deadly effects of ROS, causative agents of lipid peroxidation and other diseases either alone or in combination with diet/nutritional supplements.

In vivo anticoccidial activity of berberine [18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a) quinolizinium]--an isoquinoline alkaloid present in the root bark of Berberis lycium.

Coccidiosis, caused by various Eimeria species, is a major parasitic disease in chicken. However the increasing resistance of these parasites to currently used anticoccidial drugs has stimulated the search for new methods of control. As part of this effort we investigated the root bark of Berberis lycium (barberry) as a potential source of compounds with anticoccidial activity. In the present study anticoccidial activity of different solvent extracts of the root bark of B. lycium and berberine was evaluated in vivo using broiler chicken. Results of the study demonstrated equipotent efficacy of pure berberine in comparison to that of standard drug amprolium on the basis of reduction in coccidian oocyst output, body weight gain of chicken and feed conversion ratio. Among the extracts crude methanolic extract showed highest anticoccidial activity tested at 300 mg/kg body weight which could be due to the presence of alcohol-soluble active ingredients in root bark of B. lycium. Toxicological studies revealed that B. lycium extracts as well as berberine were not lethal up to dosage of 2000 mg/kg body weight. LD(50) was not determined as mortalities were not recorded in any of the five groups of chicken. From the present study it can be concluded that root bark of B. lycium has the immense potential to contribute to the control of coccidian parasites of chicken. Our results corroborate the use of berberine for treatment of severe diarrhoea, amoebiasis and intestinal infections and could justify its use in folk medicine for treatment of haemorrhagic dysentery.

Cuminum cyminum and Carum carvi: An update.

Cuminum cyminum and Carum carvi are the sources of cumin and caraway seeds respectively, which have been used since antiquity for the treatment of various indications in traditional healing systems in wide geographical areas. Cumin and caraway seeds are rich sources of essential oils and have been actively researched for their chemical composition and biological activities. In recent times (especially during the last 3 years) considerable progress has been made regarding validation of their acclaimed medicinal attributes by extensive experimental studies. In this attempt many novel bioactivities have been revealed. This review highlights the significance of cumin and caraway as potential source of diverse natural products and their medicinal applications.

Involvement of P-glycoprotein and CYP 3A4 in the enhancement of etoposide bioavailability by a piperine analogue.

Etoposide, a semi-synthetic derivative of podophyllotoxin, is widely used anticancer agent. Etoposide presents low bioavailability with wide inter-, and intra-patient variability after oral dosing. In an earlier study a piperine analogue, namely, 4-ethyl 5-(3, 4-methylenedioxyphenyl)-2E,4E-pentadienoic acid piperidide (PA-1), was shown to cause 2.32-fold enhancement of the absolute bioavailability of co-dosed etoposide in mice. In the present investigation a mechanistic evaluation was undertaken using various in vitro and animal-derived models. In everted rat gut sac studies PA-1 enhanced mucosal uptake of the drug while it inhibited efflux of Rh-123, a P-glycoprotein substrate from serosal-to-mucosal direction. In a single pass in situ perfusion experiment PA-1 significantly reduced the intestinal exsorption rate, exsorption clearance and the total plasma clearance of etoposide. On the other hand PA-1 did not alter the passive diffusion pattern of the drug in PAMPA assay. PA-1 was inhibitory to NADPH-assisted deethylation and demethylation reactions catalyzed by erythromycin N-demethylase, 7-methoxycoumarin-O-demethylase (MOCD) and ethoxyresorufin-O-deethylase (EROD). PA-1 was not cytotoxic to mucosal membrane and showed no adverse effect in acute toxicity determination. The results suggested that PA-1-mediated enhancement in the oral bioavailability of etoposide could possibly be due to its ability to modify P-gp/CYP 3A4 mediated drug disposition mechanisms.

Possible role of macrophages induced by an irridoid glycoside (RLJ-NE-299A) in host defense mechanism.

In order to explore the possible role of macrophages and other necessary immune competent (T and B) cells in the modulation of immune responses, an attempt was made to study the immunomodulatory effect of an irridoid glycoside (RLJ-NE-299A) isolated from the roots of Picrorhiza kurroa. Both in vitro and in vivo studies were used to evaluate the effect of RLJ-NE-299A on humoral, cellular, and phagocytic activity of macrophages. The data obtained in the present study showed that RLJ-NE-299A significantly increased sheep red blood cell (SRBC) and induced antibody (IgM and IgG) titer and delayed type hypersensitivity (DTH) reaction in mice. Besides augmenting the humoral and cell-mediated immune response, it induced macrophage phagocytosis and stimulated cytokine-induced macrophage activation and nitric oxide (NO) production, which resulted in a high degree of protection against Candida albicans and Salmonella typhimurium infections. Flow cytometric analysis indicated the enhanced expression of co-stimulatory surface molecules CD80 and CD86. The ability of RLJ-NE-299A to upregulate these cell surface antigens involved in antigen presentation may provide an explanation for the increased T-cell mediated immunity involving macrophages. Taken together this in vitro and in vivo preclinical data suggests that RLJ-NE-299A acts as an effective immunomodulator specifically to improve macrophage function during infections. The effects of this agent on these cells at concentrations relevant to in vivo therapy support its immunopharmacologic application to modify cellular immunity.

A novel sarsasapogenin glycoside from Asparagus racemosus elicits protective immune responses against HBsAg.

The aim of the present investigation was to evaluate the adjuvant potential of a novel sarsasapogenin glycoside (immunoside) isolated from Asparagus racemosus in combination with hepatitis B surface antigen (HBsAg). Various in vitro and animal derived protocols were used to determine the response of immunoside adjuvanted with HBsAg and the results were compared with alum adjuvanted with HBsAg. Several biomarkers such as antibody titre (IgG, IgG1/IgG2a) were measured in mice sera. Cell proliferation, cytokines (IL-2, IFN-γ and IL-4), and lymphocyte sub-populations (CD4/CD8, CD3 and CD19) were determined in splenocytes from mice administered subcutaneously with test substances. In these cells CD4/CD8 derived IFN-γ release was also determined. Macrophage preparations were used for the determination of IL-12, IFN-γ and nitrite content. Seroconversion potential was compared with a standard vaccine. Acute safety evaluation of immunoside was done in mice. Effect of immunoside on red blood cell haemolysis was determined. The results have suggested that immunoside potentially enhanced anti-HBsAg immune response via augmenting Th1/Th2 response in a dose dependent manner.

Simultaneous determination of etoposide and a piperine analogue (PA-1) by UPLC-qTOF-MS: evidence that PA-1 enhances the oral bioavailability of etoposide in mice.

In the present investigation, a UPLC-qTOF-MS/MS method has been developed for the simultaneous determination of etoposide and a piperine analogue, namely, 4-ethyl 5-(3,4-methylenedioxyphenyl)-2E,4E-pentadienoic acid piperidide (PA-1). The analytes were separated on a reverse phase C18 column using methanol-water (72:28, v/v) mobile phase with a flow rate of 250 microL/min. The qTOF-MS was operated under multiple reaction monitoring mode using electro-spray ionization (ESI) technique with positive ion polarity. The major product ions for etoposide and PA-1 were at m/z 185.1350 and 164.1581, respectively. The recovery of the analytes from mouse plasma was optimized using solid phase extraction technique. The total run time was 6 min and the elution of etoposide and PA-1 occurred at 1.24 and 2.84 min, respectively. The calibration curves of etoposide as well as PA-1 were linear over the concentration range of 2-1000 ng/mL (r(2), 0.9829), and 1-1000 ng/mL (r(2), 0.9989), respectively. For etoposide intra-assay and inter-assay accuracy in terms of % bias was in between -7.65 to +6.26, and -7.83 to +5.99, respectively. For PA-1 intra-assay and inter-assay accuracy in terms of % bias was in between -7.01 to +9.10, and -7.36 to +6.71, respectively. The lower limit of quantitation for etoposide and PA-1 were 2.0 and 1.0 ng/mL, respectively. Analytes were stable under various conditions (in autosampler, during freeze-thaw, at room temperature, and under deep-freeze conditions). The method was used for a pharmacokinetic study which showed that PA-1 enhanced the oral bioavailability of etoposide in mice by 2.32-fold.

Modulation of P-glycoprotein ATPase activity by some phytoconstituents.

In the present investigation 16 phytoconstituents, which are active moieties found in several medicinal herbs, have been evaluated for their P-glycoprotein (P-gp) stimulation/inhibition profiles using a P-gp-dependent ATPase assay in rat jejunal membrane (in vitro). Acteoside, agnuside, catechin, chlorogenic acid, picroside -II and santonin showed an inhibitory effect. Negundoside, picroside -I and oleanolic acid caused a stimulatory effect. Andrographolide, apocyanin, berberine, glycyrrhizin, magniferin and piperine produced a biphasic response (stimulation at low concentration and inhibition at high concentration). The results suggested that a possible interaction of these phytoconstituents at the level of P-gp, could be an important parameter in determining their role in several key pharmacodynamic events.

Anti-histaminic potential of fractions from Hedranthera barteri [(Hook F.) Pichon] root in laboratory rodents.

Hedranthera barteri, anti-inflammatory activity, anti-nociceptive activity, anti-histaminic activityL. (HB) is used in the treatment of painful conditions and oedema amongst its folkloric use. The hexane and ethyl acetate fractions of the root of H. barteri were investigated for anti-nociceptive and antiinflammatory properties and probable mechanism of action. Hot plate, tail flick, formalin-induced oedema and acetic acidinduced writhing tests were employed to investigate the anti-nociceptive activity while the anti-inflammatory activity was investigated using carrageenan-induced paw oedema. Anti-histaminic potential of HB root extracts on the rat peritoneal mast cells (RPMCs) was explored through pectrofluorometric method. The root was screened for its phytochemical components. The HB root contains alkaloids,cardenolides and saponins. HXHBR exhibited higher anti-inflammatory potentials (P <0.001). HXHBR dose-dependently (P <0.01) reduced the histamine release from the rat peritoneal mast cells which is comparable with a standard anti-histaminic drug, ketotifen. These results showed that EAHBR and HXHBR possess anti-nociceptive and anti-inflammatory activities, and suggested its mechanism of action through the inhibition of histamine, an inflammatory mediator, usually released during the early phase of allergic responses and chronic phase of inflammatory pain. Flavonoids, alkaloids and/or saponins present in HB root may be responsible for its anti-nociceptive and anti-inflammatory properties.

Pharmacokinetic interaction of some antitubercular drugs with caraway: implications in the enhancement of drug bioavailability.

This study deals with the pharmacokinetic interaction of selected anti-TB drugs with a natural product (CC-1a) derived from caraway (Carum carvi, L.) seed. CC-1a, chemically standardized butanolic fraction, enhanced the plasma levels of rifampicin, pyrazinamide, and isoniazid in Wistar rat, resulting in increased bioavailability indices (C(max) and AUC) of the drugs. Moreover, a 40% reduced dose regimen of these drugs, which additionally contained CC-1a, was equivalent in terms of C(max) and AUC to a normal dose regimen. A permeation-enhancing property of CC-1a across small intestinal absorptive surface was found to be a contributing factor in its bioavailability enhancing profile.

In vivo genotoxicity evaluation of a plant based antiarthritic and anticancer therapeutic agent Boswelic acids in rodents.

The genotoxic potential of anti-inflammatory/anti-arthritic and anticancer plant based drug molecule Boswelic acids (BA) was studied by in vivo system. Systematic literature survey revealed that studies on the genotoxicity of BA are not available. Although reports on genotoxicity of Boswellia serrata dry extract and modified 3-O-acetyl-11-keto-beta-boswelic acid are available and these studies were conducted in in vitro systems. The earlier general toxicity study of BA has been conducted by us, revealed it to be non toxic. The genotoxicity was carried out in Wistar rats using different cytogenetic assay system-abnormalities viz. chromosomal aberrations; sperm morphology, micronuclei and comet assays. Six groups of animals, each comprised of five rats, were taken for each study. Group1-4 received BA at 125, 250, 500 and 1000 mg/kg p.o., respectively prepared as 2% gum acacia suspension, fifth group received a positive control cyclophosphamide (CP) 40 mg/kg p.o. or metronedazole (MTZ) 130 mg/kg p.o. or mercuric chloride (HgCl(2)) 0.864 mg/kg p.o. (as per the experiment requirement) whereas the sixth group kept as vehicle control. The results on the bases of the data obtained revealed that BA is quite safe as it did not show any genotoxicity at any dose level up to 1000 mg/kg. The positive controls used in different experiments showed highly significant abnormal cytogenetic changes in comparison to the control group.

Sub-acute toxicity evaluation of an aqueous extract of Labisia pumila, a Malaysian herb.

Labisia pumila (Myrsinaceae), is a popular herb among the women in Malaysia known locally as "Kacip Fatimah". Recently many nutraceutical products containing the powdered or extracted parts of the plant have become available for women's health care. However no evaluation of the effect of the repeated dosing of any herbal product of this plant had been undertaken prior to a 28-day sub-acute study presented in this report. The results showed that a dose of 50mg/kg of an aqueous extract of L. pumila corresponded to no-adverse-effect-level (NOAEL), whereas higher doses were associated with some toxicity concerns.

Anti-allergic effects of herbal product from Allium cepa (bulb).

Allium cepa (Family Liliaceae) is a reputed Indian medicinal herb that is prescribed as an effective remedy for several ailments in the Ayurvedic system of medicine. The aim of this study was to evaluate its efficacy against various events responsible for Type I allergic reactions. A herbal fraction (ALC-02) from A. cepa (bulb) inhibited histamine release and attenuated intracellular calcium levels in Compound 48/80-induced rat peritoneal mast cells. It also prevented Compound 48/80-mediated systemic anaphylaxis while lowering histamine levels in plasma. ALC-02 suppressed carrageenan-induced rat paw edema. It inhibited eosinophil peroxidase activity and protein content in bronchoalveolar lavage fluid (BALF) of ovalbumin-challenged mice. In this experiment ALC-02 also caused a substantial reduction in lipid peroxidation in BALF/lung tissue and augmented superoxide dismutase activity in lung tissue. ALC-02 suppressed erythrocytic lysis caused by Triton X-100. A significant quenching of 1,1-diphenyl-2-picrylhydrazyl radical by ALC-02 was observed. The results have shown a promising anti-allergic profile of ALC-02 that could be attributed to its potential antihistaminic, anti-inflammatory, and antioxidant activities.

Anti-histaminic, anti-inflammatory and bronchorelaxant activities of 2, 7-dimethyl-3-nitro-4H pyrido [1,2-a] pyrimidine-4-one.

An immunopharmacological profile of 2, 7-dimethyl-3-nitro-4H pyrido [1,2-a] pyrimidine-4-one (P-I) has been investigated using in vitro and in vivo models representing various features of Type I allergy. P-I prevented compound 48/80-mediated histamine release from rat peritoneal mast cells. A promising anti-inflammatory activity of P-I was evident in active paw anaphylaxis (mice) and carragenan-induced paw edema (rat). P-I inhibited eosonophil accumulation and eosinophil peroxidase activity in bronchoalveolar lavage fluid from ovalbumin challenged balb/c mice: in these animals blood levels of IL-5, and CD4+ T cells also remained attenuated. A promising bronchorelaxant effect of P-I was observed in histamine-contracted guinea pig tracheal chain via its antagonism to H1 receptor. These findings were compared with some known compounds (ketotifen, cetirizine and promethazine). The anti-histaminic, anti-inflammatory and bronchorelaxant activities of P-I has been discussed in context with its potential profile as an anti-allergic and anti-asthmatic agent.

The gastric ulcer protective effect of boswellic acids, a leukotriene inhibitor from Boswellia serrata, in rats.

Aim of the study is to evaluate the anti-ulcer efficacy of the boswellic acids (BA), a triterpenoid known as anti-inflammatory/anti-arthritic agent, which is in clinical use. The reason for the study is that, the known non-steroidal anti-inflammatory drugs (NSAIDs) are full of side effects especially ulceration which is at the top. BA, although, used as an anti-arthritic agent yet it is not only devoid of ulcer production but protective also. The activity evaluation was done by the following universally accepted animal models viz., pyloric ligation, ethanol-HCl, acetylsalicylic acid, indomethacin and cold restrained stress-induced ulceration in rats. Results of the present study revealed that BA possess a dose dependent antiulcer effect against different experimental models. It showed different degree of inhibition of the ulcer score towards different ulcerogenic agents. The ulcer score against various ulcer inducing agents viz., pyloric ligation, ethanol/HCl, (acute and chronic) acetylsalicylic acid, indomethacin and cold restraint stress, was inhibited by 39%, 38%, 51%, 31%, 37% and 42% respectively at 250mg/kg. From the data it is concluded that BA inhibited ulcer production non-specifically in all the experimental models, whereby, it is not possible to propose a single specific mechanism. Nevertheless it is possible that BA might be acting by increasing the gastric mucosal resistance and local synthesis of cytoprotective prostaglandins and inhibiting the leukotriene synthesis.

Simultaneous high-performance liquid chromatographic determination of Cedrus deodara active constituents and their pharmacokinetic profile in mice.

A specific and sensitive high-performance liquid chromatographic (HPLC) method with photodiode-array (PDA) ultraviolet detection was developed for the simultaneous determination of three bioactive constituents of Cedrus deodara namely wikstromol, matairesinol and dibenzylbutyrolactol in mouse plasma. In solid-phase extraction (SPE) these constituents were successfully separated using a C18 column by isocratic elution using acetonitrile:water containing hexanesulphonic acid, 32:68 (v/v). The flow rate was set at 1ml/min and detector wavelength at 225nm. Good linearity (r2>0.999) was observed over the studied range of 0.015-5.0microg/ml for wikstromol and 0.030-5.0microg/ml for matairesinol and dibenzylbutyrolactol. The CV values of intra-day precision for wikstromol, matairesinol and dibenzylbutyrolactol were in between 1.8-6.9, 1.7-4.9 and 1.6-4.2% and values of inter-day precision were in between 10.4-12.2, 9.7-11 and 10-11.2%, respectively. The extraction recoveries at low to high concentration were greater than 98, 83 and 87% for each analyte, respectively. The LOQ for wikstromol was 0.015microg/ml and for both matairesinol and dibenzylbutyrolactol it was 0.030microg/ml. The developed method was used to determine the pharmacokinetics of the three analytes in mice after intraperitoneal administration of CD-3.

Boswellic acids: A leukotriene inhibitor also effective through topical application in inflammatory disorders.

Boswellic acids (BA), a natural mixture isolated from oleo gum resin of Boswellia serrata comprised of four major pentacyclic triterpene acids: beta-boswellic acid (the most abundant), 3-acteyl-beta-boswellic acid, 11-keto-beta-boswellic acid, and 3-acetyl-11-keto-beta-boswellic acid, is reported to be effective as anti-inflammatory, immunomodulatory, anti-tumor, anti-asthmatic and in Chron's disease. It inhibits pro-inflammatory mediators in the body, specifically leukotrienes via inhibition of 5-lipoxygenase, the key enzyme of leukotriene synthesis, is the scientifically proved mechanism for its anti-inflammatory/anti-arthritic activity. All previous work on BA for its biological activity has been done through the systemic application but no pre-clinical data reported for its anti-inflammatory activity by topical application. We here by report anti-inflammatory activity of BA through this route by applying different acute and chronic models of inflammation i.e., arachidonic acid and croton oil-induced mouse ear edema, carrageenan-induced rats paw edema and adjuvant-induced developing arthritis in rats. The results of the study revealed that the effect observed through this route is in accordance to the study conducted with the systemic route, thus establishing that BA when used through topical application is as effective as through the systemic route.

Reversal of hepatotoxin-induced pre-fibrogenic events by Emblica officinalis--a histological study.

Efficacy of a herbal product of E. officinalis (fruit) (EO) has been evaluated against carbon tetrachloride (CCl4) and thioacetamide (TAA) induced changes in rat liver. Chronic treatment of CCl4 and TAA revealed abnormal histopathology indicative of pre-fibrogenic events. EO reversed such alterations with significant regenerative changes suggestive of its preventive role in prefibrogenesis of liver.