Characterization of Turpentine nanoemulsion and assessment of its antibiofilm potential against methicillin-resistant Staphylococcus aureus.

Turpentine essential oil (TEO) is a commercially available product having application as food additive, due to its ethno-botanical and ethnopharmacological properties. In the present study, we performed chemical composition of TEO by Gas Chromatography-Mass Spectrometry (GC-MS). Further, TEO was nanoemulsified, encapsulated and characterized by droplet size, PDI, Zeta potential and transmittance. The obtained turpentine nanoemulsion (TNE) was investigated for its antibacterial and antibiofilm potentiality against methicillin-resistant Staphylococcus aureus (MRSA), a model biofilm-forming microorganism. Small micellar TEO nanoparticles were succesfully formed with a mean droplet size ranging from 22.52 to 26.54 nm. Thermodynamic stability studies revealed homogeneous dispersion of the droplets size confirming the stability of TNEs. The developed nano-emulsions displayed two fold enhanced antagonistic activity against S. aureus in comparison with TEOs, with minimum inhibitory concentration (MIC) values at 0.039% (v/v) against MRSA. Additionally, TNEs displayed potent antibiofilm activity against MRSA strains with percent biofilm disruption of around 70.83%. Findings from this study validates the phytomedicinal significance of turpentine nanoemulsions and envisage its exploration as a natural and cost-effective strategy against bacterial biofilms in medical and industrial sectors.

Modulation of volatile release and antimicrobial properties of pink pepper essential oil by microencapsulation in single- and double-layer structured matrices.

The bioactivity of essential oils applied in foods to act as natural preservatives can be reduced due to interactions with other components of the food matrix. Microencapsulation can help to increase the functionality of these compounds. In addition, the electrostatic interaction between proteins and polysaccharides can result in double-layered encapsulating structures, ensuring greater protection to essential oils than using only protein as surface active agent. In this work, pink pepper essential oil was microencapsulated by spray drying of single-layer emulsions, stabilized by soy protein isolate (SPI), and of double-layer emulsions, stabilized by soy protein isolate/high methoxyl pectin (SPI/HMP). Pink pepper essential oil showed predominance of α-pinene, ß-pinene, ß-mircene, δ-3-carene, d-limonene, and germacrene D. Compared to SPI microcapsules, SPI/HMP microcapsules better preserved the total volatile content identified in pure oil, showed less water adsorption during storage at relative humidity ≥75% and improved antimicrobial properties. When stored for 20 days (25 °C/RH = 52.8%), both microcapsules allowed more gradual release of volatiles compared with non-encapsulated oil. Microencapsulation by spray drying did not have negative effects on the antioxidant activity of the encapsulated oil, as the microcapsules showed similar results to the non-encapsulated oil, around 11 µg Trolox/mg of oil. After storage, however, the non-encapsulated oil showed greater losses of its antioxidant activity due to higher rates of volatile release. In the in vitro antimicrobial activity assay, both microcapsules inhibited growth of Staphylococcus aureus, Bacillus subtilis, Listeria monocytogenes and Listeria innocua, although no inhibition was observed against Gram-negative bacteria. When added in milk, both microcapsules reduced bacterial growth, whereas non-encapsulated oil showed no satisfactory inhibition. Faster reduction of microbial growth in milk was observed for SPI/HMP microcapsules. Inhibition results were better for skim milk than for whole milk, suggesting that the interaction of essential oil with other lipids present in milk decreased its bioactivity. Microencapsulation positively affected the functionality of pink pepper essential oil, highlighting its potential for application as a natural preservative in food products.

Effect of solvents on apically extruded debris and irrigant during root canal retreatment using reciprocating instruments.

AIM: To evaluate the effect of several solvents on the weight of apically extruded debris and irrigant during retreatment using a novel agar gel model. METHODS: Orange oil, turpentine oil and chloroform were used as solvents. Eighty single straight-rooted extracted human mandibular premolar teeth with a single root canal were divided into four groups (n = 20). All specimens were root canal-filled and weighed prior to their insertion into a prepared 1.5% agar gel model. The mean initial weights were measured by subtracting the weight of the specimen from the weight of the test apparatus and recorded. Following the removal of the coronal 4 mm of root filling, the test solvent was applied onto the root filling. No solvent was used in the control group. A Reciproc R25 instrument was used to remove the root filling in all groups. Apically extruded debris and test solvent were collected during retreatment procedures. The mean weights of apically extruded debris and irrigant were calculated by subtracting the mean initial weights from the weights of test apparatus without the Teflon tape and the specimen following the retreatment procedures. Data were statistically analysed using one-way analysis of variance. RESULTS: Use of solvents resulted in significantly less extruded debris and irrigant compared to the control group (P < 0.05). Chloroform extruded significantly more debris than orange oil and turpentine oil (P < 0.05). CONCLUSION: Use of solvents during root filling removal was associated with less apically extruded debris and irrigant when compared to no solvent.

Inflammation inhibits the expression of phosphoenolpyruvate carboxykinase in liver and adipose tissue.

Inhibition of adipocyte triglyceride biosynthesis is required for fatty acid mobilization during inflammation. Triglyceride biosynthesis requires glycerol 3-phosphate and phosphoenolpyruvate carboxykinase (PEPCK) plays a key role. We demonstrate that LPS, zymosan, and TNF-α decrease PEPCK in liver and fat. Turpentine decreases PEPCK in liver, but not in fat. The LPS-induced decrease in PEPCK does not occur in TLR4 deficient animals, indicating that this receptor is required. The LPS-induced decrease in hepatic PEPCK does not occur in TNF receptor/IL-1 receptor knockout mice, but occurs in fat, indicating that TNF-α/IL-1 is essential for the decrease in liver but not fat. In 3T3-L1 adipocytes TNF-α, IL-1, IL-6, and IFNγ inhibit PEPCK indicating that there are multiple pathways by which PEPCK is decreased in adipocytes. The binding of PPARγ and RXRα to the PPARγ response element in the PEPCK promoter is markedly decreased in adipose tissue nuclear extracts from LPS treated animals. Lipopolysaccharide and zymosan reduce PPARγ and RXRα expression in fat, suggesting that a decrease in PPARγ and RXRα accounts for the decrease in PEPCK. Thus, there are multiple cytokine pathways by which inflammation inhibits PEPCK expression in adipose tissue which could contribute to the increased mobilization of fatty acids during inflammation.

Therapeutic effect of Linum usitatissimum (flaxseed/linseed) fixed oil on acute and chronic arthritic models in albino rats.

The present study was undertaken to assess the activity/anti-inflammatory potential of Linum usitatissimum fixed oil against castor oil-induced diarrhoea, turpentine oil-induced joint oedema, formaldehyde and Complete Freund's Adjuvant (CFA)-induced arthritis in Wistar albino rats. The oil intraperitoneally, significantly inhibited the castor oil-induced diarrhoea and turpentine oil-induced exudative joint oedema in a dose-dependent manner. Significant inhibitory effect of L. usitatissimum fixed oil was observed in formaldehyde-induced proliferative global oedematous arthritis when given intraperitoneally, with significant checking of the serum glutamic oxaloacetic acid transaminase and serum glutamic pyruvic acid transaminase. Further, L. usitatissimum fixed oil showed a significant dose-dependent protective effect against CFA-induced arthritis as well. Secondary lesions produced by CFA due to a delayed hypersensitivity reaction were also reduced in a significant manner. Anti-inflammatory activity of L. usitatissimum fixed oil can be attributed to the presence of alpha linolenic acid (57.38%, an omega-3 fatty acid, 18:3, n-3) having dual inhibitory effect on arachidonate metabolism resulting in suppressed production of proinflammatory n-6 eicosanoids (PGE(2), LTB(4)) and diminished vascular permeability. These observations suggest possible therapeutic potential of L. usitatissimum fixed oil in inflammatory disorders like rheumatoid arthritis.

The essential oil of turpentine and its major volatile fraction (alpha- and beta-pinenes): a review.

This paper provides a summary review of the major biological features concerning the essential oil of turpentine, its origin and use in traditional and modern medicine. More precisely, the safety of this volatile fraction to human health, and the medical, biological and environmental effects of the two major compounds of this fraction (alpha- and beta-pinenes) have been discussed.

Interleukin-6 regulates the zinc transporter Zip14 in liver and contributes to the hypozincemia of the acute-phase response.

Infection and inflammation produce systemic responses that include hypozincemia and hypoferremia. The latter involves regulation of the iron transporter ferroportin 1 by hepcidin. The mechanism of reduced plasma zinc is not known. Transcripts of the two zinc transporter gene families (ZnT and Zip) were screened for regulation in mouse liver after turpentine-induced inflammation and LPS administration. Zip14 mRNA was the transporter transcript most up-regulated by inflammation and LPS. IL-6 knockout (IL-6(-/-)) mice did not exhibit either hypozincemia or the induction of Zip14 with turpentine inflammation. However, in IL-6(-/-) mice, LPS produced a milder hypozincemic response but no Zip14 induction. Northern analysis showed Zip14 up-regulation was specific for the liver, with one major transcript. Immunohistochemistry, using an antibody to an extracellular Zip14 epitope, showed both LPS and turpentine increased abundance of Zip14 at the plasma membrane of hepatocytes. IL-6 produced increased expression of Zip14 in primary hepatocytes cultures and localization of the protein to the plasma membrane. Transfection of mZip14 cDNA into human embryonic kidney cells increased zinc uptake as measured by both a fluorescent probe for free Zn(2+) and (65)Zn accumulation, as well as by metallothionein mRNA induction, all indicating that Zip14 functions as a zinc importer. Zip14 was localized in plasma membrane of the transfected cells. These in vivo and in vitro experiments demonstrate that Zip14 expression is up-regulated through IL-6, and that this zinc transporter most likely plays a major role in the mechanism responsible for hypozincemia that accompanies the acute-phase response to inflammation and infection.

Antioxidant and analgesic activities of turpentine of Pinus nigra Arn. subsp. pallsiana (Lamb.) Holmboe.

The aim of this study is to examine possible antioxidant and analgesic activities of turpentine exudes from Pinus nigra Arn. subsp. pallsiana (Lamb.) Holmboe (TPN). Total antioxidant activity, reducing power, superoxide anion radical scavenging, free radical scavenging, metal chelating, and hydrogen peroxide scavenging activities were studied. The total antioxidant activity increased with the increasing amount of extracts (100, 300, and 500 microg) added to linoleic acid emulsion. All of the doses of TPN showed higher antioxidant activity than alpha-tocopherol. The samples showed 49, 70, and 91% inhibition on peroxidation of linoleic acid emulsion, respectively. On the other hand, the 300 microg of alpha-tocopherol showed 40% inhibition on peroxidation of linoleic acid emulsion. There is correlation between antioxidant activity and the reducing power, superoxide anion radical scavenging, free radical scavenging, metal chelating, and hydrogen peroxide scavenging activities. Like antioxidant activity, the reducing power, superoxide anion radical scavenging, free radical scavenging, metal chelating, and hydrogen peroxide scavenging activities of TPN depending on concentration and increasing with increased concentration of TPN. These properties may be the major reasons for the inhibition of lipid peroxidation. The results obtained in the present study indicate that the TPN has a potential source of natural antioxidant. In addition, analgesic effect of TPN was investigated in present study and TPN had strong analgesic effect. The analgesic effect of TPN compared with metamizol as a standard analgesic compound.

Larvicidal effects of mineral turpentine, low aromatic white spirits, aqueous extracts of Cassia alata, and aqueous extracts, ethanolic extracts and essential oil of betel leaf (Piper betle) on Chrysomya megacephala.

BACKGROUND: Many methods have been employed, with variable success, in the treatment of cutaneous myiasis caused by Chrysomya species. AIMS: Experiment 1: to assess the larvicidal effect of mineral turpentine (MT) and the main ingredient of MT, low aromatic white spirits (LAWS), on Chrysomya megacephala larvae in vitro. Experiment 2: to assess the larvicidal effects of aqueous extracts of winged senna (Cassia alata), and aqueous extracts, ethanolic extracts and essential oil of betel leaf (Piper betle). METHODS: In experiment 1, two samples of LAWS were obtained from two industrialists (samples 1 and 2). Adult flies of C. megacephala were bred in the insectory of the Department of Parasitology, Faculty of Medicine, University of Colombo. Petri dishes were prepared with pads of cotton wool. These cotton pads were soaked separately in MT, LAWS samples 1 and 2, and normal saline as a control. Ten larvae were placed in each Petri dish. The activity of the larvae was observed and recorded half-hourly. MT and the two samples of LAWS were analyzed by chromatography. In experiment 2, volatile essential oil of betel was prepared using a standard steam distillation process. An ethanolic extract of betel was obtained after boiling the crushed leaf with water, and mixing the stock with ethanol. Betel oil dilutions of 1-4% were prepared using 1% Tween 80 (v/v aq) as a solvent, with 0.05 g/100 mL sodium lauryl sulphate (as stabilizer) and 0.01 g/100 mL methyl paraben (as a preservative). Cotton wool swabs soaked in 1, 2, 3 and 4% essential oil of betel in 1% Tween 80 (v/v aq) prepared as above, 1, 2, 3 and 4% ethanolic extract of betel, 50 and 25% aqueous extract of C. alata, and 50 and 25% aqueous extract of betel were placed in separate Petri dishes. Ten larvae were placed in each Petri dish. 1% Tween 80 solvent with the stabilizer and the preservative, but without betel essential oil, was used as a negative control and MT was used as a positive control. Larval motility was assessed as before. RESULTS: MT and the two LAWS samples killed the larvae in vitro within 4 h. Chromatography showed more unidentified constituents in MT than in pure LAWS, indicating additional substances in MT. The 4 and 3% preparations of the essential oil of betel were effective in killing 100% of the larvae of Chrysomya within 3 h 30 min. The 2% extract of betel essential oil killed 96.7% of larvae in 4 h. Ethanolic and aqueous extracts of betel, the aqueous extract of C. alata, normal saline and the Tween 80 solvent were not larvicidal. CONCLUSIONS: MT and LAWS, the main ingredient of MT, were effective in killing Chrysomya larvae. Essential oil obtained from betel leaves also showed a dose-dependent larvicidal effect on Chrysomya larvae. This natural product may be effective in the treatment of wound myiasis.

Effects of Melilotus officinalis on acute inflammation.

Our study investigated the effects of Melilotus officinalis L. extract, containing 0.25% coumarin, on acute inflammation induced with oil of turpentine in male rabbits. The results were compared with those from a group treated with hydrocortisone sodium hemisuccinate and one injected with coumarin before inflammation was induced. The effects were evaluated by measuring serum citrulline, a test of in vitro phagocytosis, total leukocyte count and differential leukocyte count expressed as a percentage. M. officinalis had antiinflammatory effects because it reduced the activation of circulating phagocytes and lowered citrulline production. These properties were similar to those of hydrocortisone sodium hemisuccinate and coumarin. In the bone marrow acute phase response, M. officinalis had an inhibitory action that was lower than that of hydrocortisone sodium hemisuccinate and similar to coumarin.

Enhancing effect of essential oils on the penetration of 5-fluorouracil through rat skin.

Three essential oils as penetration enhancers for 5-fluorouracil (5-FU) were studied using excised rat skin. The oils used were eucalyptus, peppermint and turpentine. Azone was used for comparison. The enhancing effect of the oils was found to be less than that of azone, but all the oils used enhanced the permeation of 5-FU. Eucalyptus oil was found to be the most active, causing about 60 fold increase, while peppermint and turpentine caused 46 and 28 fold increase, respectively. Eucalyptus oil was further studied by grading it into 5 fractions according to difference in boiling points. It was found that their activities increased as their boiling point increased. With all enhancers increased partition coefficients were observed but the diffusion coefficient values obtained were comparatively higher. The mode of action of these accelerants may be described by combined processes of partition and diffusion, the diffusion process being dominant.

Effects of turpentine oil pretreatment on beta-blocker pharmacokinetic parameters in rats.

Turpentine oil treatment (0.2 mL kg-1, s.c.) was used to increase the plasma concentration of alpha 1-acid glycoprotein (0.13 mg mL-1 in control rats) to 1.72 mg mL-1 after 2 days, and allow assessment of its effects on the pharmacokinetics and stereoselective binding of three beta-blockers. Racemates (5 mg kg-1) were administered intravenously to control and turpentine oil-pretreated rats and the plasma concentrations were determined up to 90 min. Stereoselective analysis showed the apparent distribution volume and the area under plasma concentration-time curves (AUC) of R-(+)-propranolol to be, respectively, one-quarter and twice those of the S-(-)-enantiomer and differences in pharmacokinetic parameters between the two were magnified by turpentine oil pretreatment. Pharmacokinetic parameters of oxprenolol enantiomers were essentially similar for the controls but after turpentine oil pretreatment, a higher affinity of the R-(+)-enantiomer for plasma was observed. Acebutolol enantiomers behaved non-stereospecifically throughout. These results were consistent with predictions from the in-vitro stereospecific binding properties of these agents to purified rat alpha 1-acid glycoprotein.

Receptive properties of myelinated primary afferents innervating the inflamed urinary bladder of the cat.

1. The present study has investigated the receptive properties of myelinated mechanoreceptive primary afferents innervating the inflamed urinary bladder of the cat. In 15 experiments, 20 units were recorded from the dorsal and 3 from the ventral root S2. Before inflammation the afferents had no resting activity and responded consistently to increases of intravesical pressure evoked by isotonic distension or isovolumetric contractions. All units were studied before and after the onset of an acute inflammation induced by intraluminal injection of mustard (1-2.5%) or turpentine oil (50%), which are known to induce an acute cystitis. 2. Eleven out of 14 units tested with mustard oil and 5/9 units tested with turpentine oil were activated at short latency. The response could not be explained by a concomitant increase of intraluminal pressure resulting from the intravesical injection of the irritant. This suggests that a large proportion of mechanosensitive afferents has an additional chemosensitivity. 3. After removal of the irritants and with empty bladder, all afferent units exhibited irregular ongoing activity with intermittent high-frequency bursts. Such ongoing activity was entirely absent in myelinated afferents supplying the noninflamed bladder. The median rate of ongoing activity was significantly higher after mustard oil (1.65 imp/s) than after turpentine oil treatment (0.05 imp/s) 1 h after chemical stimulation. Post-hoc analysis revealed that afferents that developed high levels of ongoing activity had steeper stimulus response functions to changes of intravesical pressure before inflammation. 4. The stimulus-response function of vesical afferents changed characteristically in the inflamed bladder. Within 30 min of mustard oil treatment, the responses of some units to bladder filling was transiently enhanced, but later the units desensitized to this stimulus. However, there was no significant change of the stimulus-response function of six afferents studied before and for 2 h after induction of the inflammation. By contrast, the afferents exhibited significant increases of their mechanosensitivity after turpentine treatment. 5. In conclusion, sacral myelinated mechanoreceptive afferents supplying the urinary bladder are chemosensitive. Their receptive properties change rather uniformly when the tissue becomes inflamed, and, on the basis of these changes, it is not possible to differentiate subpopulations of primary afferents. The induction of ongoing activity and the increased responses to intravesical pressure stimuli in the inflamed organ, together with central changes that may occur under these conditions, are probably responsible for changes of bladder motility and sensations that arise during cystitis.

Glucocorticoid status affects the response of rat striatal dopamine D2 receptors to hyperthermia and turpentine treatment.

The influence of glucocorticoid status of the rats (absence of glucocorticoid hormones achieved by adrenalectomy and substitution by dexamethasone 5 mg/kg b.w.) on the response of striatal dopamine-D2 receptors to environmentally induced hyperthermia and treatment with a local inflammatory agent turpentine (1 ml/kg b.w.) was studied. Both stress situations affected the density of D2 receptors in opposite directions. While hyperthermia led to an increase, turpentine treatment reduced Bmax value. The changes in the D2 receptor binding affinity were expressed as a certain decrement, which was statistically significant in sham-adrenalectomized hyperthermic animals and in both turpentine treated groups (sham-operated and adrenalectomized) administered dexamethasone. The absence of glucocorticoids in the circulation caused an elevation of Kd values in the control and both stressed groups and dexamethasone attenuated these changes. Dexamethasone also attenuated stress-related alterations of Bmax, as well as Kd increase upon adrenalectomy in control and hyperthermic animals. These results corroborate the evidence on the role of D2 receptors in thermoregulation and stress and demonstrate the significance of glucocorticoid hormones in the control of these processes.

Enhancement of the elicitation phase of the murine contact hypersensitivity response by prior exposure to local ultraviolet radiation.

Local exposure of the sites of elicitation (ears) of contact hypersensitivity (CHS) in mice to relatively modest doses of ultraviolet radiation (FS40 sunlamps) daily for 4 days prior to challenge in previously immunized animals results in significant enhancement of the CHS response as measured by 24-h swelling. This effect was seen in both C3H/HeJ and A/J mice and with the use of both trinitrochlorobenzene and dinitrofluorobenzene as contact sensitizers. Doses of PUVA (parenteral administration of 8-methoxypsoralen followed by exposure to UVA, 320-400 nm, radiation) up to that which can systemically suppress the induction of CHS failed to affect the elicitation of CHS. Irritation of the sites of elicitation of CHS by applications of turpentine also failed to affect the CHS elicitation reaction. Enhancement of the CHS elicitation response by local ultraviolet radiation exposure prior to challenge appears to be a specific photobiologic event.

The effects of drugs on tear flow.

Systemic administration of drugs can diminish the tear flow as with timolol and atropine, or can increase the tear production as with bromhexine. Turpentine fumes (100 ppm/30 min or 30 ppm/8 h) have been shown to diminish the break up time (BUT) of tears by diminishing the lipid layer of the precorneal film. Ointment and drugs suspended in oil also diminish BUT, not only do they dry the corneal and conjunctival epithelium (judged by the rose bengal score) but also diminish the outflow as judged by the tear river dye dilution test in both normal people and those with keratoconjunctivitis sicca. Benzalconium chloride in concentrations used as a preservative and cocaine have also been shown to reduce BUT.

Effect of inhaling medication vapors from a colds preparation on murine pulmonary bacterial defense systems.

The usefulness of murine pulmonary defense systems as a means for assessing potential toxicity was further confirmed. Recent investigations had indicated that the effects of a chemical agent on mucociliary transport and phagocytic function comprised a sensitive indicator. As one example of such an application, because of the wide usage of proprietary colds preparations containing volatile oils, as well as the use of such oils in various industries, it is important to reassess the potential of such preparations for toxicity when new test systems become available. Mice and rats were exposed to vapors of camphor, menthol, eucalyptol, and turpentine, as contained in a commonly used colds preparation, for 4 and 8 hr prior to challenge with aerosols of radiolabeled Staphylococcus aureus. The exposure system simulated conditions present when the colds preparation is vaporized according to directions for use. Rates of pulmonary bacterial transport and inactivation, as well as of phagocytic ingestion, were determined following exposure to the colds preparation. Each of these rates was unchanged by the treatment. Thus, in this biological system exposure to therapeutic levels of the colds preparation did not impair mucociliary or phagocytic function.