Excipient effect on antimicrobial activity of Cinnamon (Cinnamomum zelylanicum album) oil, Thyme (Thymus vulgaris) oil and Ajowan (Trachyspermum ammi) oil

Background:Many herbal essential oils are potential antimicrobials but their pharmaceutical utility is restricted due to a lack of suitable excipients to mollify their dermatotoxicity and irritant property,and outcome of their therapeutic use may vary with different diluents used.Methods:Effect of 16 diluents(dimethyl sulfoxide,liquid paraffin,glycerine,oils of mustard,sunflower,rice bran,palm,groundnut,olive,coconut,sesame,avocado,jojoba,castor,linseed and soybean)was assessed on antimicrobial activity of 2%cinnamon(Cinnamomum zelylanicum album),thyme(Thymus vulgaris)and ajowan(Trachyspermum ammi)oils using agar well diffusion assay.The effect of excipients was evaluated on six Candida albicans,five Escherichia coli,four Acinetobacter lwoffii,two strains each of Staphylococcus aureus,Enterobacter agglomerans,and Enterococcus faecium and one strain each of Acinetobacter calcoaceticus,Escherichia fergusonii,Klebsiella oxytoca,K.pneumoniae ssp.pneumoniae,Leclercia adecarboxylata,Paenibacillus amylolyticus,Proteus mirabilis,P.vulgaris,Pseudomonas aeruginosa,Raoultella terrigena,Staphylococcus capitis ssp.capitis,S.chromogenes,S.epidermidis,S.warneri and Streptococcus pyogenes.Results:Thyme oil(2%)maintained it antimicrobial activity on dilution in dimethyl sulfoxide and glycerine,and ajowan oil(2%)completely lost its antibacterial activity in all diluents except dimethyl sulfoxide.However,cinnamon oil partially lost its antimicrobial activity upon dilution in glycerine,vegetable,and mineral oils in comparison to dimethyl sulfoxide.Olive oil was the best vegetable oil,almost comparable to dimethyl sulfoxide and castor oil was the worst diluent for maintaining antimicrobial activity of cinnamon oil.Conclusion:The study indicated the non-suitability of vegetable oils for pharmaceutical formulations of essential oils except olive oil for dilution of cinnamon oil and glycerol for thyme oil to replace dimethyl sulfoxide as diluent.

Novel virulence factor dupA of Helicobacter pylori as an important risk determinant for disease manifestation:An overview

Helicobacter pylori(H.pylori)is a microaerophilic,Gram-negative,human gastric pathogen found usually in the mucous lining of stomach.It infects more than 50%of the world’s population and leads to gastroduodenal diseases.The outcome of disease depends on mainly three factors:Host genetics,environment and bacterial factors.Among these,bacterial virulence factors such as cagA,vacA are well known for their role in disease outcomes.However,based on the global epidemiological results,none of the bacterial virulence(gene)factors was found to be associated with particular diseases like duodenal ulcer(DU)in all populations.Hence,substantial importance has been provided for research in strain-specific genes outside the cag pathogenicity island,especially genes located within the plasticity regions.dupA found within the plasticity regions was first demonstrated in 2005 and was proposed for duodenal ulcer development and reduced risk of gastric cancer in certain geographical regions.Due to the discrepancies in report from different parts of the world in DU development related to H.pylori virulence factor,dupA became an interesting area of research in elucidating the role of this gene in the disease progression.In this review,we shed light on the detailed information available on the polymorphisms in dupA and their clinical relevance.We have critically appraised several pertinent studies on dupA and discussed their merits and shortcomings.This review also highlights dupA gene as an important biomarker for DU in certain populations.

In-vitro and In-vivo Anti-trypanosomal Activity of Terminalia chebula Retz Dried Fruits

African trypanosomosis had caused lots of havocs to both humans and animals over a century with successes and failure in curtailing it. This study was aimed at screening medicinal plant, Terminalia chebula dried fruits against Trypanosoma evansi for trypanocidal activity. Twenty grams of powdered Terminalia chebula dried fruits was cold extracted with methanol. Obtained MPE （methanolic plant extract） was in vitro tested against Trypanosoma brucei （1 × 10^6 trypanosomes/mL of the medium in each ELISA plate wells） at concentrations （250～1,000 μg/mL） on Vero cells grown in DMEM （Debecco＇s Modified Eagle Medium） in appropriate conditions for trypanocidal activity. In-vitro cytotoxicity test of MPE of Terminalia chebula was conducted on Vero cells grown in DMEM. In-vivo assay for trypanocidal activity, each mouse was inoculated with 1 × 10^4/mL of trypanosomes and treated （48 h post inoculation） with MPE of Terminalia chebula at concentrations （12.5, 25, 50, 100 and 200 mg/kg body weight） were administered at dose rate of 100 BL per mouse via intraperitoneal route to different groups of mice, 6 mice per concentration. In-vitro cytotoxicity test was done on Veto cells at concentrations （1.58～100 μg/mL） of MPE of Terminalia chebula. Results of in-vitro trypanocidal activity varied from immobilization, reduction and to the killing of the trypanosomes. At 250 μg/mL ofMPE ofTerminalia chebula dried fruits, there was significant trypanocidal activity at 4 h of incubation and trypanosomes were not detected in corresponding ELISA plate wells at 5 h of incubation, which was statistically equivalent to reference drug, diminazine aceturate （50 μL/mL） at 4 h of incubation. Results of in-vivo trypanocidal activity revealed that at concentrations （l 2.5～25 mg/kg body weight） of MPE of Terrninalia chebula, mice in these groups survived for 6 days. While at 50 and 100 to 200 mg/kg body weight, mice in these groups survived up to 7 and 8 days, respectively. In-vitro cytotoxicity test showed that all concentrations of MPE of Terminalia chenula and diminazine aceturate were cytotoxic to cells except at 1.56 μL/mL and 6.25 μL/mL. In conclusion, MPE of Terminalia chebula dried fruits possessed trypanocidal compounds. Further study （bioassay-guided purification） is required to know the full potential of Terrninalia chebula as future trypanocide candidate.

Interaction of <i>Helicobacter pylori</i>Cell Membrane with Non-Esterified Cholesterol and Other Steroids

Helicobacter pylori performs the unique action of assimilating exogenous non-esterified cholesterol into its cell membrane. This bacterium aggressively incorporates non-esterified cholesterol into the membrane, induces its glucosylation, and uses both non-esterified cholesterol and glucosylated cholesterols as membrane lipid compositions. The reason for this assimilation of non-esterified cholesterol into the cell membrane of H. pylori has eluded investigators for many years. Recent hypotheses posit that the sterol-uptake and sterol-glucosylation contribute to the survival of H. pylori cells in different ways. The incorporation of the non-esterified cholesterol into the cell membrane fortifies the resistance of H. pylori against the antibacterial actions of phosphatidylcholines, antibiotics, and bile salts. In parallel, the glucosylation of the non-esterified cholesterol incorporated into the cell membrane serves H. pylori in two ways. First, it helps the bacterium evade host immune responses, such as phagocytosis by macrophages and activation of antigen-specific T cells. Second, it detoxifies sterols fatal to the bacterium via a novel action of sterol glucosylation recently described in another report from our group. The reluctance of H. pylori to absorb esterified cholesterol remains unexplained. A recent study by our group has demonstrated that the phosphatidylethanolamine (PE) in the outer membrane of H. pylori serves as a steroid-binding lipid the incorporation of non-esterified cholesterol into the membrane. We have also discovered that the myristic acid (C14:0) molecule attached to the PE of this bacterium plays an important role in the selective binding of non-esterified cholesterol but not esterified cholesterol.

Therapeutic Activity of Partially Purified Fractions of Emblica officinalis （Syn, Phyllanthus embfica） Dried Fruits against Trypanosoma evansi

Emblica officinalis （E. oJficinalis） dried fruits were evaluated for its antitrypanosomal activity and cytotoxic effects. Vero cell line maintained in DMEM （Dubecco＇s Modified Eagle Medium） and incubated with Trypanosoma evansi for more than 12 h. MPE was added to the Vero cell culture medium at different concentrations （250-1,000 μg/mL） with trypanosomes concentration （1 × 106 trypanosomes/mL in each ELISA plate well） and incubated at appropriate conditions for 72 h. In-vitro cytotoxieity of MPE of E. officinalis was determined on Vero cells at concentrations （（1.56-100 ~tg/mL）. Acute toxicity and in-vivo infectivity tests were done in mice. Obtained MPE ofE. officinalis underwent process of purification via column chromatography, preparative chromatography and HPLC （higher performance liquid chromatography） with bioassay at different strata on Alsever＇s medium. In-vivo assay for trypanocidal activity, MPE and PPFs （partially purified fractions） of E. officinalis with two sets of mice, each mouse was inoculated with 1 × 104/mL oftrypanosomes and treated （48 h post inoculation） at concentrations （12.5, 25, 50, 100 and 200 mg/kg body weight） were administered at dose rate of 100 [tL per mouse via intraperitoneal route （in treating parassitemic mice） to different groups of mice, 6 mice per concentration. HPLC of partially purified fractions ofE. officinalis was carried out with mobile phase ofacetonitdle： water （40：60） in gradient mode. In vitro, MPE induced immobilization and killing of the parasites in concentration-time dependent manner. Significant reduction of trypanosomes counts from concentration of 250μg/mL and complete killing of trypanosomes at 5th hour of observation, which was statistically equivalent to 4th hour of Diminazine Aceturate （Berenil）, standard reference drug used. HPLC of the partially purified fractions revealed two major prominent peaks at retention time of 1-4 min. In vivo, both MPE and PPFs of test material did prolong lives of mice by 6-9 days but could not cure them. At concentration of 2,000 kg/kg body weight of MPE in acute test, all mice survived. For in-vivo infectivity test, mice injected with immobilized trypanosomes developed parasitemia and died while, the other group survived. MPE, PPFs and Diminazine Aceturate were toxic to Vero cells at all concentrations exception of 1.56, 1.56-3.13 and 1.56-6.25 μg/mL, respectively. From this report, PPFs ofE. officinalis dried fruits demonstrated potential pathway for a new development oftrypanocide in near future if additional investigations are put in place.

Curcumin as a potential therapeutic candidate for Helicobacter pylori associated diseases

Curcumin, a yellow pigment and principal polyphenolic Curcuminoid obtained from the turmeric rhizome Curcuma longa, is commonly used as a food-coloring agent. Studies suggest that curcumin has a wide range of beneficial properties e.g., anti-inflammatory, antioxidant, anti-cancer, anti-proliferative, anti-fungal and anti-microbial. These pleiotropic activities prompted several research groups to elucidate the role of curcumin in Helicobacter pylori(H. pylori) infection. This is the first review with this heading where we discussed regarding the role of curcumin as an anti-H. pylori agent along with its potential in other gastrointestinal diseases. Based on several in vitro, early cell culture, animal research and few pre-clinical trials, curcumin projected as a potential therapeutic candidate against H. pylori mediated gastric pathogenesis. This review sheds light on the anti-H. pylori effects of curcumin in different models with meticulous emphasis on its anti-oxidant, anti-inflammatory and anti-carcinogenic effects as well as some critical signaling and effecter molecules. Remarkably, non-toxic molecule curcumin fulfills the characteristics for an ideal chemopreventive agent against H. pylori mediated gastric carcinogenesis but the foremost challenge is to obtain the optimum therapeutic levels of curcumin, due to its low solubility and poor bioavailability. Further, we have discussed about the possibilities for improving its efficacy and bioavailability. Lastly, we concluded with the anticipation that in near future curcumin may be used to develop a therapeutic drug against H. pylori mediated gastric ailments through improved formulation or delivery systems, facilitating its enhanced absorption and cellular uptake.

Indistinguishable cellular changes in gastric mucosa between Helicobacter pylori infected asymptomatic tribal and duodenal ulcer patients

AIM： To investigate the changing pattern of different histological parameters occurring in the stomach tissue of Helicobacter pylori （H pylori） infected tribal populations and duodenal ulcer patients among ethnic Bengalis and correlation of the genotypes of H pylori with different histological parameters. METHODS： One hundred and twelve adult individuals were enrolled into this study between 2002 and 2004. Among them, 72 had clinical features of duodenal ulcer （DU） from ethnic Bengali population and 40 were asymptomatic ethnic tribals. Endoscopic gastric biopsy samples were processed for histology, genotyping and rapid urease test. Histologically, haematoxylin and eosin staining was applied to assess the pathomorphological changes and a modified Giemsa staining was used for better detection of Hpylori. For intestinal metaplasia, special stainings, i.e. Alcian blue periodic acid-Schiff and high iron diamine-Alcian blue staining, were performed. PCR was performed on bacterial DNA to characterize the presence or absence of virulence-associated genes, like cagA, and distribution of different alleles of vacA and iceA. RESULTS： Intraglandular neutrophil infiltration, a hallmark of activity of gastritis, was present in 34 （94%） of tribals （TRs） and 42 （84%） of DU individuals infected with H pylori. Lymphoid follicles and aggregates, which are important landmarks in H pylori infection, were positive amongst 15 （41%） of TRs and 20 （40%） of DU subjects. Atrophic changes were observed in 60% and 27.7%, respectively, among DU cases and tribals （P 〉 0.003）. Metaplastic changes were detected in low numbers in both groups. Moderate to severe density distribution of Hpylori in the gastric mucosa was 63% among TRs, whereas it was 62% in DU subjects. There were no significant differences in the distribution of virulence-associated genes like cagA, vacA and iceA of H pylori strains carried by these two populations. CONCLUSION： Our study showed almost similar distribution of inflammatory cells among asymptomatic tribals and DU Bengali patients. Interestingly, the tribal population are free from any clinical symptoms despite evidence of active histologic gastritis and infection with Hpylori strains carrying similar virulence markers as of strains isolated from patients with DU. There was an increased cellular response, especially in terms of neutrophil infiltration, but much lower risk of developing atrophy and metaplastic changes among the tribal population.