Novel approach for the inhibition of Helicobacter pylori contamination in yogurt using selected probiotics combined with eugenol and cinnamaldehyde nanoemulsions.

Different strains of probiotics were screened in vitro to select the one with the highest anti-H. pylori activity. Three nanoemulsions of eugenol, cinnamaldehyde and their mixture were fabricated and tested also in vitro against the same pathogen. The selected probiotic strains, the nanoemulsion mixture and their combination were imbedded in a lab-manufactured yogurt which is deliberately contaminated with 6.0 log cfu/g H. pylori during manufacture. The inhibitory activity of all treatments on the growth of H. pylori and the other microorganisms in yogurt was evaluated during 21 days. Combining the selected probiotic strains with the nanoemulsion mixture in the contaminated yogurt reduced the count of H. pylori by 3.9 log cycle. The nanoemulsion showed lower inhibitory effect against the other microorganisms like probiotics, starter culture and total bacterial count in the tested yogurt, where their enumeration did not fall below 106 cfu/g at the end of yogurt storage period.

Basil Essential Oil and Its Nanoemulsion Mitigate Non Alcoholic Steatohepatitis in Rat Model with Special Reference to Gut Microbiota.

The present research evaluated the protective effect of basil essential oil nanoemulsion (BNO) and its parent basil essential oil (BO) towards steatohepatitis. Chemical composition of BO was assessed followed by formulation into different BNOs using the low energy spontaneous emulsification technique. An ideal formula of BNO was selected among the others based on its ultra-fine particle size (15.42 nm) and physical stability at 25-37°C, which was then tested in steatohepatitis rat model along with BO. Rats were divided into four groups, the first was fed on balanced diet (C), and the other groups were maintained on high fructose saturated fat diet deficient in choline to induce steatohepatitis, one of such groups served as control steatohepatitis (SC), the other groups received daily oral dose of BO and BNO, respectively. Microbiota (Firmicutes and Bacteroidetes) were counted in colon content and their ratio (F/B) was calculated. Liver fat, plasma lipid profile, plama interlukin-6, plasma lipopolysaccharides and plasma and colon content of lipocaline were assessed with histopathological examination of liver and colon. Results showed that the major volatile components of BO were linalool (60.9 %), eugenol (5.1 %) and eucalyptol (9.5%). SC group exhibited significant increase in liver lipids, plasma triglycerides, total cholesterol (TC), low density lipoprotein cholesterol and significant reduction in high density lipoprotein-cholesterol (HDL-C) compared to C group. Significant increase in plasma TC/HDL-C, interlukin-6, and lipocaline and F/B ratio and lipocaline in colon content were demonstrated in SC group without changes in plasma lipopolysaccharides compared to C. Histopathology of SC group showed liver fatty degeneration and fibroblasts activation while the colon demonstrated erosion and mucosal epithelium detachment. Treatment with either BNO or BO showed improvement compared to SC group. BNO was superior in reducing F/B ratio, liver lipids and histopathological changes. BO was more efficient in reducing TC, triglycerides and low density lipoprotein cholesterol. It is concluded that BO and BNO reduced the progression of nonalcoholic steatohepatitis in rat model. Gut microbiota in relation to steatohepatitis and related new therapies needs further investigations.

Hepatic Regeneration and Reno-Protection by Fish oil, Nigella sativa Oil and Combined Fish Oil/Nigella sativa Volatiles in CCl4 Treated Rats.

The aim of the present research was to investigate the effect of fish oil, crude Nigella sative oil and combined fish oil/Nigella sative volatile oil as hepato-regenerative and renal protective supplements. The oils were administered as emulsions to rat model with liver injury induced by CCl4. Plasma activities of transaminases (AST and ALT) were evaluated as liver function indicators, while plasma creatinine and urea and creatinine clearance were determined as markers of kidney function. Plasma malondialdehyde (MDA), nitrite (NO) and tumor necrosis factor-α (TNF-α) were estimated to assess the exposure to oxidative stress and subsequent inflammation. Liver fat was extracted and their fatty acids´ methyl esters were determined using gas chromatography. Results showed that plasma activities of AST and ALT were significantly higher in CCl4 control group compared to control healthy group. Plasma levels of creatinine and urea increased significantly in CCl4 control, while creatinine clearance was reduced significantly in the same group. All rat treated groups given the three oil emulsions showed improvement in liver function pointing to the initiation of liver regeneration. The combination of fish oil/Nigella sative volatiles showed the most promising regenerative activity. Oxidative stress and inflammation which were increased significantly in CCl4 control group showed improvement on administration of the three different oil emulsions. Fatty acids methyl ester of liver fat revealed that rats treated with fish oil/Nigella sative volatile oil presented the highest content of unsaturated fatty acids (45.52% ± 0.81) while fish oil showed the highest saturated fatty acids (53.28% ± 1.68). Conclusion; Oral administration of oil emulsions of native fish oil, Nigella sative crude oil and combined fish oil/Nigella sative volatile oil reduced liver and kidney injury in rat model of CCl4 through exerting anti-inflammatory and antioxidant activity. Fish oil/Nigella sative volatile oil emulsion was the most promising hepato-regenerative and reno-protective formula among the different groups.

Evaluation of the Antiproliferative Activity of Some Nanoparticulate Essential Oils Formulated in Microemulsion on Selected Human Carcinoma Cell Lines.

BACKGROUND: Essential oils (EOs) are natural volatile plant extracts that have different biological activities including antiproliferative potentials. OBJECTIVE: The current study aims at evaluating the antiproliferative activities against some cancer cell lines of six EOs in their neat oily state and in water-based microemulsions where the EOs exist as nanoparticles. The EOs included marjoram (Origanum majorana), turmeric (Curcuma longa), sweet basil (Ocimum basillicum), clove buds (Syzygium aromaticum), geranium (plargonium graviolenis), and black cumin (Nigella sativa). METHOD: GC-MS chromatographic analysis was used to reveal the chemical composition of EOs. Self-microemulsification method and oil titration method were used for the fabrication of the different microemulsions. MTT assay and IC50 determinations were used for evaluating the extent of the antiproliferative activity. RESULTS: Results indicated that geranium EO was the most active against the evaluated cancer cell lines followed by basil EO and marjoram EO. On the other hand, turmeric followed by black cumin EOs showed the least antiproliferative activity relative to the other EOs. Clove EO showed selective activity depending on the type of cancer cell lines. Formulation of these EOs in microemulsions led to the development of water-borne nanoparticles having an average particle size from 10.7nm to 18.0 nm depending on the type of EO. Re-evaluation of the antiproliferative activity of these EOs after microemulsification showed differential behavior ranging from activity enhancement to retardation relative to the original activity of each corresponding neat EO in its oily state. Potential factors that could justify the obtained results are discussed. CONCLUSION: Some EOs and their microemulsions may potentially be used as natural adjuvants to classical anti-cancer drugs.

Chemical composition and antimicrobial activity of garlic essential oils evaluated in organic solvent, emulsifying, and self-microemulsifying water based delivery systems.

The chemical composition of garlic essential oils (GEOs) extracted from two different cultivars has been characterized using GC-MS analysis. GEO that was extracted from the white-skin cultivar (WGO) had a lower percentage of the major constituents diallyl trisulfide and diallyl disulfide (45.76 and 15.63%) than purple-skin cultivar (PGO) which contained higher percentages (58.53 and 22.38%) of the same components, respectively. Evaluation of the antimicrobial activity of WGO and PGO delivered in organic solvent (isopropanol) showed dose-dependent antimicrobial activity against the tested pathogenic bacteria and fungi, especially with WGO. On the other hand, formulation of both GEOs in water-based emulsions totally suppressed the antimicrobial activity of GEO. Re-formulation of GEOs in water-based microemulsion (particle size 10.1nm) showed better antimicrobial activity than emulsions at the same concentration of GEOs. This study can assist in designing the proper water-based delivery system of GEO for application in food preservation.

Microencapsulation of Nigella sativa oleoresin by spray drying for food and nutraceutical applications.

Oleoresin of Nigella sativa L. (Black cumin) was obtained from the seeds using hexane extraction at room temperature. The oleoresin was emulsified in an aqueous solution containing gum Arabic/maltodextrin (1:1 w/w) and then encapsulated in powder form by spray drying. The characteristics of the obtained powder including moisture content, bulk density, wettability, morphology, encapsulation efficiency were evaluated. The effect of the spray drying on the chemical composition of the volatile oil fraction of N. sativa oleoresin was also evaluated using gas chromatographic-mass spectroscopic analysis. Results indicated that the encapsulation efficiency of the whole oleoresin in the powder can range from 84.2±1.5% to 96.2±0.2% depending on the conditions of extracting the surface oil from the powder. On the other hand the encapsulation efficiency of the volatile oil fraction was 86.2% ±4.7. The formulated N. sativa L. oleoresin powder can be used in the fortification of processed food and nutraceuticals.

Factors affecting the phase behavior and antimicrobial activity of carvacrol microemulsions.

The aim of this study was to investigate some factors that can contribute to the formulation of aqueous-based carvacrol microemulsion that can potentially be used in food preservation or disinfection. For this purpose the capacity of formation of carvacrol microemulsion was first revealed by studying the phase behavior of that compound in five different non-ionic microemulsion systems. Factors affecting that phase behavior like the type of non-ionic surfactant and presence of solubilization enhancers were also studied. The fully dilutable microemulsion system that can incorporate high carvacrol amount, as revealed from the phase diagrams, was chosen for the antibacterial evaluation study. The same microemulsion system was re-formulated in a cationic form by substituting the non-ionic surfactant, Tween 20 (T20) with the cationic cetylpyridinium chloride (CPC). The disc diffusion method was used to evaluate the activity of these microemulsion systems against different pathogenic bacteria. Results of the phase behavior study showed that carvacrol is a challenging phenolic compound which did not lend itself easily for solubilization in a fully dilutable non-ionic microemulsion. Incorporation of some solubilization enhancers like propylene glycol (PG) or short chain alcohols can fulfill this purpose however high surfactant/carvacrol ratio (9:1) was still required to solubilize only 1.0 wt% carvacrol in dilutable microemulsion. The antibacterial evaluation study at that concentration revealed that non-ionic carvacrol microemulsion formulated with T20 and a solubilization enhancer did not exhibit better antimicrobial activity than the same concentration of carvacrol formulated in surfactant-free aqueous solution composed of water/PG (1:1). On the other hand, the CPC-formulated carvacrol microemulsion showed significantly higher antibacterial activity than T20-formulated microemulsion. Results of the current investigation shed the light on the solubilization capacity and phase behavior of carvacrol in non-ionic microemulsion and the potential of using cationic carvacrol microemulsion in disinfection and decontamination applications.

Analysis and antibacterial activity of Nigella sativa essential oil formulated in microemulsion system.

The Essential oil (EO) of Nigella sativa (black cumin) was extracted from the crude oil and the volatile constituents were characterized using gas chromatographic analysis. The EO was formulated in water-based microemulsion system and its antibacterial activity against six pathogenic bacteria was evaluated using the agar well diffusion method. This activity was compared with two other well known biologically active natural and synthetic antimicrobials namely eugenol and Ceftriaxone(®). Results showed that N. sativa EO microemulsion was highly effective against S. aureus, B. cereus and S. typhimurium even at the lowest tested concentration of that EO in the microemulsion (100.0 µg/well). Interestingly, the EO microemulsion showed higher antibacterial activity than Ceftriaxone solution against S. typhimurium at 400.0 µg/well and almost comparable activity against E. coli at 500.0 µg/well. No activity was detected for the EO microemulsion against L. monocytogenes and P. aeruginosa. Eugenol which was also formulated in microemulsion was less effective than N. sativa EO microemulsion except against P. aeruginosa. The synthetic antibiotic (Ceftriaxone) was effective against most of the six tested bacterial strains. This work is the first report revealing the formulation of N. sativa EO in microemulsion system and investigating its antibacterial activity. The results may offer potential application of that water-based microemulsion in controlling the prevalence of some pathogenic bacteria.

Protective effect of clove oil and eugenol microemulsions on fatty liver and dyslipidemia as components of metabolic syndrome.

In the present research, the effect of clove essential oil (CO) and its major constituent, eugenol, formulated in water-based microemulsions, was studied on fatty liver and dyslipidemia in high-fructose-fed rats. Plasma and liver lipids, oxidative stress, inflammatory biomarker, and liver function were the assessed criteria. CO dispersed in water as conventional cloudy emulsion was also subjected to the same biological evaluations for comparison with the microemulsified form of this oil. Results showed that the particle size of CO microemulsion (COM) and eugenol microemulsion (EM) was 8.0 nm and 8.9 nm, respectively. Excess dilution and incubation of these microemulsions in 1.2 N HCl, that mimic stomach juice (without lipase), for 5 hours at 37 °C lead to the establishment of second population of larger particles with average diameter>100.0 nm. Biological evaluation revealed that rats of high fructose control group exhibited significant dyslipidemia, high plasma tumor necrosis factor-α, and elevated malondialdehyde. The same group of rats showed significant high liver total fat, triglycerides and cholesterol, and liver dysfunction compared to control normal rats fed balanced diet. Daily oral administration of CO conventional emulsion, COM, and EM produced significant improvement of all studied parameters. No significant change in all biochemical parameters was noticed when the groups given the different formulations were compared with each other. The study concluded that administration of CO conventional emulsion, COM, or EM produced significant improvement in fatty liver and dyslipidemia with consequent expected protection from cardiovascular diseases and other complications of fatty liver. Formulation of CO in microemulsion having particle size ∼ 8.0 nm did not enhance the protective effect compared with the same dose of CO dispersed in water as conventional macroemulsion, probably due to the ease of absorption of these bioactives in their native states. However, formulation in microemulsion provides a delivery system for oral administration of CO or eugenol in homogeneous, water-based, and thermodynamically stable dosage form during storage.

Formulation and shelf life stability of water-borne lecithin nanoparticles for potential application in dietary supplements field.

ABSTRACT The objective of the present investigation is to formulate commercial soybean lecithin as nanoparticles in solvent-free aqueous system for potential supplementary applications. A mechanical method, which involved two major steps, was used for that purpose. First, lecithin submicron particles (~ 0.5 µm) have been prepared by gradual hydration of lecithin powder using mechanical agitation. Finally, the size of these particles was further reduced to < 100 nm by using high-pressure microfluidization. The physical stability (appearance, particle size distribution, ζ-potential) and the chemical stability (lipid oxidation) of the dispersions carrying lecithin nanoparticles were assessed every 15 days during the 3-month shelf life period at two different temperatures. Results showed that the final particle size of lecithin in the freshly prepared aqueous dispersion was 79.8 ± 1.0 nm and the amount of peroxide detected was 3.5 ± 0.2 meq/kg lipid. At the end of the storage period, dispersions stored at 4°C exhibited physical and chemical stability as evident from the translucent appearance, the small change in particle size (84.1 ± 1.3 nm), and the small amount of generated peroxides (4.1 ± 0.2 meq/kg lipid). On the other hand, dispersions stored at 25°C were physically stable up to 60 days. Over that period, samples became turbid and the particle size increased to 145.0 ± 1.7 nm with a bimodal distribution pattern. This behavior was due to phospholipids (PLs) degradation and hydrolysis under acidic conditions, which proceeds faster at a relatively high temperature (25°C) than at (4°C). The outcome of this investigation may help in developing water-based dispersions carrying lecithin nanoparticles for dietary supplement of PLs.

The chemical composition and the content of volatile oil: potential factors that can contribute to the oxidative stability of Nigella sativa L. crude oil.

The crude oil of Nigella sativa L. (Black cumin) has well-known nutraceutical and pharmaceutical properties. The oil is prone to rapid oxidative deterioration because of its high content of poly-unsaturated fatty acids. In the current investigation, different cold-pressed crude oils of N. sativa were examined for their composition of fatty acids and oxidative stability. The data obtained were correlated with the chemical composition and content of volatile oils, which correspond to each crude oil. Results indicated that different crude oils have the same fatty acid composition, and linoleic acid was the major constituent (60.0-61.7%). Though, the oxidative stability index of the crude oils was different ranging from 2.5 hr to 26.9 hr, as revealed by the Rancimate test at 100°C and airflow rate of 20 ml/min. The results showed that there is a straightforward correlation between the volatile oil content and the oxidative stability of the corresponding parent crude oil. In addition, high γ-terpinene content in the constitution of volatile oil can also be another contributing factor for enhancing the oxidative stability of the crude oil. The data presented here draw attention to some important factors that may guide the nutraceutical and food supplement processors in their screening for the highest quality of Nigella crude oils with maximum health benefits.

Anti-cancer properties of Nigella spp. essential oils and their major constituents, thymoquinone and beta-elemene.

Essential oils are the volatile fraction of aromatic and medicinal plants after extraction by steam or water distillation. They have been used for their pharmaceutical potential since early times, and even now are still subject to a great deal of attention, as is clear from the increasing number of publications each year on this subject. This review presents both fundamental and recent studies concerned with the role of Nigella species essential oils and their major constituents, thymoquinone and beta-elemene, as potential chemotherapeutic and chemopreventive anti-cancer agents. The mechanism of action and the factors which determine the concentrations of these major constituents in the essential oil are also reviewed.

Pharmaceutical and therapeutic potentials of essential oils and their individual volatile constituents: a review.

Essential oils and their volatile constituents are used widely to prevent and treat human disease. The possible role and mode of action of these natural products is discussed with regard to the prevention and treatment of cancer, cardiovascular diseases including atherosclerosis and thrombosis, as well as their bioactivity as antibacterial, antiviral, antioxidants and antidiabetic agents. Their application as natural skin penetration enhancers for transdermal drug delivery and the therapeutic properties of essential oils in aroma and massage therapy will also be outlined.

Antifungal activity of peppermint and sweet basil essential oils and their major aroma constituents on some plant pathogenic fungi from the vapor phase.

The vapors of peppermint oil and two of its major constituents (menthol and menthone), and sweet basil oil and two of its major constituents (linalool and eugenol), were tested against Sclerotinia sclerotiorum (Lib.), Rhizopus stolonifer (Ehrenb. exFr.) Vuill and Mucor sp. (Fisher) in a closed system. These fungi cause deterioration and heavy decay of peach fruit during marketing, shipping and storage. The essential oils, their major individual aroma constituents and blends of the major individual constituents at different ratios inhibited the growth of the fungi in a dose-dependent manner. Menthol was found to be the individual aroma constituent responsible for the antifungal properties of peppermint essential oil, while menthone alone did not show any effect at all doses. In the case of basil oil, linalool alone showed a moderate antifungal activity while eugenol showed no activity at all. Mixing the two components in a ratio similar to their concentrations in the original oil was found to enhance the antifungal properties of basil oil indicating a synergistic effect.