Therapeutic Potential of Clove Essential Oil in Diabetes: Modulation of Pro-Inflammatory Mediators, Oxidative Stress and Metabolic Enzyme Activities.

Type 1 diabetes is characterized by insulin deficiency due to the destruction of pancreatic ß cells, leading to hyperglycemia, which in turn induces vascular complications. In the current study, we investigated the effect of intraperitoneal administration of clove essential oil (CEO: 20 mg/kg body weight) on certain oxidative stress and glucose metabolism enzymes, as well as the expression of proinflammatory mediators. Administration of CEO to diabetic rats showed a significant decline in blood glucose levels, total cholesterol, and xanthine oxidase, compared to the streptozotocin group. Furthermore, these treated rats elicited a notable attenuation in the levels of lipid peroxides, and thiols groups in both liver and brain tissues. The activities of antioxidant and metabolic enzymes were reverted to normality in diabetic upon CEO administration. In addition to its protective effects on red blood cell hemolysis, CEO is a potent α-amylase inhibitor with an IC50 =298.0±2.75 µg/mL. Also, treatment of diabetic rats with CEO significantly reduced the iNOS expression in the spleen. Our data showed that CEO has potential beneficial effects on diabetes, which can possibly prevent the pathogenesis of diabetic micro- and macrovascular complications.

Clove Essential Oil and Its Main Constituent, Eugenol, as Potential Natural Antifungals against Candida spp. Alone or in Combination with Other Antimycotics Due to Synergistic Interactions.

The occurrence of candidiasis, including superficial infections, has recently increased dramatically, especially in immunocompromised patients. Their treatment is often ineffective due to the resistance of yeasts to antimycotics. Therefore, there is a need to search for new antifungals. The aim of this study was to determine the antifungal effect of clove essential oil (CEO) and eugenol (EUG) towards both reference and clinical Candida spp. strains isolated from the oral cavity of patients with hematological malignancies, and to investigate their mode of action and the interactions in combination with the selected antimycotics. These studies were performed using the broth microdilution method, tests with sorbitol and ergosterol, and a checkerboard technique, respectively. The CEO and EUG showed activity against all Candida strains with a minimal inhibitory concentration (MIC) in the range of 0.25-2 mg/mL. It was also found that both natural products bind to ergosterol in the yeast cell membrane. Moreover, the interactions between CEO and EUG with several antimycotics-cetylpyridinium chloride, chlorhexidine, silver nitrate and triclosan-showed synergistic or additive effects in combination, except nystatin. This study confirms that the studied compounds appear to be a very promising group of phytopharmaceuticals used topically in the treatment of superficial candidiasis. However, this requires further studies in vivo.

Investigating Antiarthritic Potential of Nanostructured Clove Oil (Syzygium aromaticum) in FCA-Induced Arthritic Rats: Pharmaceutical Action and Delivery Strategies.

The combined application of clove oil in a lipid nanocarrier opens a promising avenue for bone and joints therapy. In this study, we successfully developed a tunable controlled-release lipid platform for the efficient delivery of clove oil (CO) for the treatment of rheumatoid arthritis (RA). The ultra-small nanostructured lipid carriers co-loaded with CO (CONCs) were developed through an aqueous titration method followed by microfluidization. The CONCs appeared to be spherical (particle size of 120 nm), stable (zeta potential of -27 mV), and entrapped efficiently (84.5%). In toluene:acetone:glacial acetic acid (90:9:1 percent v/v/v) solvent systems, high-performance thin layer chromatography (HPTLC) analysis revealed the primary components in CO as eugenol (RF = 0.58). The CONCs greatly increased the therapeutic impact of CO in both in vitro and in vivo biological tests, which was further supported by excellent antiarthritic action. The CONC had an antiarthritic activity that was slightly higher than neat CO and slightly lower than standard, according to our data. The improved formulation inhibited serum lysosomal enzymes and proinflammatory cytokines while also improving hind leg function. This study provides a proof of concept to treat RA with a new strategy utilizing essential oils via nanodelivery.

Novel Clove Essential Oil Nanoemulgel Tailored by Taguchi's Model and Scaffold-Based Nanofibers: Phytopharmaceuticals with Promising Potential as Cyclooxygenase-2 Inhibitors in External Inflammation.

PURPOSE: Clove essential oil is a phytochemical possessing a vast array of biological activities. Nevertheless, fabricating nano topical delivery systems targeted to augment the anti-inflammatory activity of the oil has not been investigated so far. Accordingly, in this study, controlled release nanoparticulate systems, namely nanoemulgel and nanofibers (NFs), of the oil were developed to achieve such goal. METHODS: The nanoemulsion was incorporated in the hydrogel matrix of mixed biopolymers - chitosan, guar gum and gum acacia - to formulate nanoemulsion-based nanoemulgel. Taguchi's model was adopted to evaluate the effect of independently controlled parameters, namely, the concentration of chitosan (X1), guar gum (X2), and gum acacia (X3) on different dependently measured parameters. Additionally, the nanoemulsion-based NFs were prepared by the electrospinning technique using polyvinyl alcohol (PVA) polymer. Extensive in vitro, ex vivo and in vivo evaluations of the aforementioned formulae were conducted. RESULTS: Both Fourier transform-infrared spectroscopy (FT-IR) and differential scanning calorimetry (DSC) established the complete dispersion of the nanoemulsion in the polymeric matrices of the prepared nanoemulgel and NFs. The ex vivo skin permeation data of clove essential oil from the prepared formulations showed that NFs can sustain its penetration through the skin comparably with nanoemulgel. Topical treatment with NFs (once application) and nanoemulgel (twice application) evoked a marvelous in vivo anti-inflammatory activity against croton oil-induced mouse skin inflammation model when compared with pure clove essential oil along with relatively higher efficacy of medicated NFs than that of medicated nanoemulgel. Such prominent anti-inflammatory activity was affirmed by histopathological and immunohistochemical examinations. CONCLUSION: These results indicated that nanoemulsion-based nanoemulgel and nanoemulsion-based NFs could be introduced to the phytomedicine field as promising topical delivery systems for effective treatment of inflammatory diseases instead of nonsteroidal anti-inflammatory drugs that possess adverse effects.

Alternative sanitizers to paraformaldehyde for incubation of fertile eggs.

The aim of this study was to evaluate an ethanolic extract of propolis and clove essential oil as a substitute for paraformaldehyde for the sanitation of fertile eggs. In total, 1,800 hatching eggs (from 40-week-old CPK [Pesadão Vermelho] breeder hens) were randomly distributed among the treatments (grain alcohol, clove essential oil, ethanolic extract of propolis, and paraformaldehyde). Spraying was the application method for all treatments except for paraformaldehyde, for which fumigation was used. The experimental design was a randomized block design with 4 treatments. Analysis of the incubation parameters was based on 6 replications per treatment. The egg weight loss was lower in the eggs treated with ethanolic extract of propolis (8.59 ± 3.34%) than in the eggs treated with grain alcohol (13.40 ± 2.87%), clove essential oil (12.96 ± 3.33%), and paraformaldehyde (13.05 ± 3.24%). The hatchability of the fertile eggs (51.39 ± 5.81%) and the hatchability of the set eggs (44.74 ± 6.79%) were negatively affected by the application of ethanolic extract of propolis. Late mortality of eggs treated was higher than early mortality in the grain alcohol (12.14 ± 4.72%; 2.86 ± 3.30%), clove essential oil (4.60 ± 5.95%; 3.03 ± 3.50%), and ethanolic extract of propolis (36.63 ± 6.60%, 11.98 ± 4.30%) treatments. The eggs treated with clove essential oil (67.90 ± 1.87%), paraformaldehyde (67.80 ± 1.85%), or grain alcohol (67.50 ± 1.92%) presented chick yields as expected. However, despite the ideal yield of eggs treated with ethanolic extract of propolis (69.25 ± 1.68%), its application at the concentration used in the present research is not recommended. Clove essential oil, when sprayed on fertile eggs as a sanitizing agent, did not differ from paraformaldehyde in relation to hatchery performance parameters.

Short communication: Evaluation of the efficacy of novel disbudding methods for dairy calves.

The objectives of this study were to (1) evaluate whether cryoablation or the administration of clove oil was as efficacious as cautery disbudding at preventing horn growth, and (2) evaluate whether the efficacy of cautery disbudding is affected by removing or leaving the horn bud tissue intact after disbudding of dairy calves. At approximately 4 d of age (4.0 ± 0.88 d of age, mean ± SD), 265 dairy heifer calves from 3 dairy farms (farm 1: n = 129 calves; farm 2: n = 109 calves; farm 3: n = 27 calves) were disbudded. Each calf had 1 of 4 treatments randomly assigned to each horn bud: (1) clove oil (0.5 mL) administered subcutaneously under the horn bud (CLOV, n = 135 buds); (2) a liquid nitrogen-filled probe applied to the horn bud area for 30 s (CRYO, n = 134 buds); (3) cautery disbudding using an electric hot-iron and the horn bud removed (BUDOFF, n = 130); or (4) cautery disbudding and the horn bud tissue left intact (BUDON, n = 131). Calves were assessed for signs of infection at the disbudding site frequently within the first month after disbudding. At approximately 6 mo of age (6 ± 2.2 mo, mean ± SD) calves were assessed for scur or horn growth. The disbudding procedures were considered successful if no scur or horn development was observed. Within the first month, 12% of disbudding wounds showed some indication of infection, such as pus, exudate, or swelling; of the infected buds, 52% were associated with the BUDON treatment, 27% with CLOV, 25% with BUDOFF, and 2% with CRYO treatments. At 6 mo of age, BUDOFF was the most effective method of preventing horn growth and CRYO was the least efficacious [mean percentage of success: BUDOFF: 100% (95% CI: 97.7-100.0); CRYO: 1% (95% CI: 0.2-5.3)]. Injecting clove oil under the horn bud was 87% (95% CI: 80.6-92.5) successful. Not removing the horn bud tissue after cautery disbudding reduced the efficacy of this method by 9% [91% success (95% CI: 83.8-95.7)]; moreover, this method was associated with more infection at the site of disbudding. It appears as though the clove oil treatment could be used as an alternative to cautery disbudding of dairy calves; however, further research is needed to evaluate the tissue damage and associated pain caused by clove oil and to refine this technique (i.e., administration methods to improve efficacy) before it could be considered an alternative to cautery.

Clove Bud Oil Modulates Pathogenicity Phenotypes of the Opportunistic Human Pathogen Pseudomonas aeruginosa.

Earlier studies from our laboratory have demonstrated that clove bud oil (CBO) attenuates expression of certain virulence factors of Pseudomonas aeruginosa PAO1. Here, we probe more deeply into the effect of CBO on four pseudomonal proteases - elastase A, elastase B, protease IV and alkaline protease - each known to play key roles in disease pathogenesis. CBO inhibited the activity of these proteases present in the bacterial culture supernatant. Zymography studies indicated that these proteases can activate host matrix metalloproteases (MMPs) to establish infection, through conversion of pro-MMP-2 to active MMP-2. PAO1 is a predominant pathogen in burn wound infections and we show the modulatory effect of CBO on MMPs in an in vitro model of burn injury. Furthermore, CBO induced dose-dependent neutrophil extracellular trap formation in human neutrophils. CBO also increased the survival of C. elegans infected with PAO1, establishing an anti-infective role in a whole animal model of pathogenesis. LC-MS/MS analysis indicated that CBO treatment elicited a significant reduction of signalling molecules (Acyl-Homoserine-Lactone) involved in quorum sensing regulation. Our observations demonstrate that CBO attenuates key virulence mechanisms of this important human pathogen, while concomitantly enhancing host innate immunomodulatory functions, with potential implications for topical therapy against antibiotic-resistant infections.

Effectiveness of topical clove oil on symptomatic treatment of chronic pruritus.

BACKGROUND: Pruritus is a frequent symptom in the general population and in many skin and systemic diseases. Its frequency demonstrates a high burden and an impaired quality of life. AIM: Assessment of the efficacy of topically applied clove oil alone in the treatment of chronic pruritus. METHODS: Fifty selected subjects diagnosed with chronic pruritus due to either hepatic, renal, or diabetic causes were studied and divided into two groups of 25 patients each. Group I patients were instructed to hydrate the skin and then apply topical clove oil while group II patients applied petrolatum topically by hand; this application was done on the areas of pruritus, twice daily and for 2 weeks. The severity of the itch was assessed and compared before and after the study by 5-D itch scale. The results were analyzed by SPSS. Statistical methods such as descriptive analysis, independent-samples t-test, paired-samples t-test, and chi-square were employed. RESULTS: There was a significant improvement regarding all studied individual parameters (5-D itch scale) for the clove oil users with no significance among petrolatum users. Comparison of 5-D itch scale total score between patients of group I and patients of group II favored the improvement following the use of clove oil than using placebo (P value>.05). CONCLUSION: The topical treatment of chronic pruritus with clove oils is effective, easy to use, safe, cheap, and more acceptable for whom topical and systemic treatments tend to be irritating, contraindicated, or less well tolerated.

Protective potency of clove oil and its transcriptional down-regulation of Aeromonas sobria virulence genes in African catfish (Clarias gariepinus L.).

Disease episodes of fish caused by Aeromonas species are moved to the top list of limiting problems worldwide. The present study was planned to verify the in vitro antibacterial activities as well as the in vivo potential values of clove oil and ciprofloxacin against Aeromonas sobria in African catfish (Clarias gariepinus). The in vitro phenotypic virulence activities and the successful amplification of aerolysin and hemolysin genes in the precisely identified A. sobria strain were predictive for its virulence. In the in vivo assay, virulence of A. sobria strain was fully demonstrated based on constituent mRNA expression profile of tested virulence genes and typical septicemia associated with high mortalities of infected fish. Apparent lower mortality rates were correlated well with both decrescent bacterial burden and significant down-regulated transcripts of representative genes in the treated groups with clove oil, followed by ciprofloxacin as a prophylactic use for 15 days (P < 0.0001); however, the essential oil apart from ciprofloxacin significantly enhanced different hematological parameters (P < 0.05). In addition, administration of antibiotic may be considered as a pronounced stress factor in the fish even when it used in the prophylactic dose. In conclusion, medicinal plants-derived essential oils provide a virtually safer alternative to chemotherapeutics on fish, consumers and ecosystems.

Clove (Syzygium aromaticum Linn) extract rich in eugenol and eugenol derivatives shows bone-preserving efficacy.

This study examined the efficacy of hydroalcoholic extract of dried clove buds, which is rich in phenolic compounds namely eugenol and eugenol derivatives (precursors of flavones, isoflavones and flavonoids), on different primary and secondary osteoporotic marker changes in an ovariectomised (OVX) rat model of osteoporosis. Female Wistar rats were randomly divided into three groups: sham-operated control (A), OVX (B) and OVX plus 50% hydroalcoholic extract of dried clove buds for 4 weeks (C). Results indicated that, compared to control, serum alkaline phosphatase (AP; 48.25%, p < 0.01), serum tartrate-resistant acid phosphatase (TRAP; 63.48%, p < 0.01), urinary calcium (14.70%, p < 0.01), urinary phosphate (50.30%, p < 0.01) and urinary creatinine (122.44%, p < 0.01) were significantly altered in OVX rats. All these altered responses were significantly restored (AP: 27.53%, p < 0.01; TRAP: 33.51%, p < 0.01; calcium: 53.15%, p < 0.01; phosphate: 27.49%, p < 0.01; creatinine: 46.40%, p < 0.01) by supplementation with hydroalcoholic extract of dried clove buds. Results of bone density, bone mineral content, bone tensile strength and histological analysis also showed similar trend of results, which supported initial observations of this study. It is proposed that hydroalcoholic extract of dried clove buds has bone-preserving efficacy against hypogonadal osteoporosis.

Aceite de clavo como anestésico para el pez Pacu (piaractus mesopotamicus) / Clove oil as an anesthetic for pacu fish (piaractus mesopotamicus)

Con el propósito de minimizar el daño causado a los peces por el uso de sustancias anestésicas, se ha evaluado, en este trabajo, la concentración más adecuada de aceite de clavo como anestésico para el pez pacu (Piaractus mesopotamicus). Fueron utilizados 40 peces, con peso promedio de 716,18 g. Los tratamientos fueron compuestos de soluciones con diferentes concentraciones de aceite de clavo, 30, 60, 90, 120 y 150 mg/L. Fueron utilizados dos acuarios de vidrio: uno con 20 litros de agua con la solución anestésica para cada tratamiento y otro con 20 litros de agua limpia, para el regreso de los animales de la condición de sedación. Se observaron de forma individual: el tiempo hasta llegar a la anestesia profunda y el tiempo de recuperación de la anestesia profunda. El diseño experimental fue completamente aleatorizado con cinco tratamientos (concentraciones de aceite de clavo) y ocho repeticiones (peces). Los tiempos hasta anestesia profunda e hasta la recuperación total de los peces fueron evaluados por regresión, con 5% de significancia. Se observó un comportamiento linear de los tiempos hasta la anestesia profunda y hasta la recuperación total de los peces a concentraciones crecientes de aceite de clavo. La concentración de 30 mg/L es suficiente para la inducción anestésica de los animales, por otra parte, las concentraciones más elevadas, hasta 150 mg/L, causaron inducciones aún más rápidas, sin pérdida en las condiciones para la recuperación de los peces(AU)

In order to minimize damage to fish caused by the use of anesthetic substances, this study was to evaluate the concentration of clove oil as an anesthetic most appropriate for pacu (Piaractus mesopotamicus). A total of 40 fishes, with average weight of 716.18 g. The treatments were: anesthetic procedures with solutions of different concentrations of clove oil, 30, 60, 90, 120 and 150 mg.L-1. We used two glass aquariums: one with 20 liters of water containing the anesthetic solution for each treatment and the other with 20 liters of clean water, the animals return to the condition of sedation. Were observed individually: the time to reach deep anesthesia and recovery time of deep anesthesia. The experimental design was completely randomized design with five treatments (concentrations of clove oil) and eight replicates (individuals). The data were submitted to regression, with 5% significance level. There was a linear time of anesthesia and return of fish to increasing concentrations of clove oil, and 30 mg.L-1 is sufficient for the induction of the animals, but higher concentrations of up to 150 mg.L-1, still show inductions faster, without loss in time and conditions for the recovery of fish(AU)

Clove oil cream: a new effective treatment for chronic anal fissure.

OBJECTIVE: Anal fissure is a common painful condition affecting the anal canal and causes considerable morbidity and reduction in quality of life. Surgical treatment has been associated with a degree of incontinence in up to 30% of patients. This study discussed the results of clove oil 1% cream in healing of chronic anal fissure. METHOD: A single-blind randomized comparative trial was setup to compare traditional treatment with stool softeners and lignocaine cream 5% against clove oil 1% cream for 6 weeks. RESULTS: 55 patients were included in this study, 30 patients in clove oil group and 25 patients in control group. Healing had occurred in 60% of patients in clove oil group and in 12% of patients in the control group after 3-month follow up (P < 0.001). Patients in clove oil group showed significant reduction in resting anal pressure and almost all other anorectal manometric pressures compared with patients in control group. CONCLUSION: Topical application of clove oil cream demonstrated a significant beneficial effect when applied to patients suffering from chronic anal fissure.

Eugenol and thymol, alone or in combination, induce morphological alterations in the envelope of Candida albicans.

The envelope of Candida albicans, with its outermost array of macromolecules protruding towards the environment, is pivotal to the expression of major virulence factors such as adhesiveness, and the morphological transition to hyphal form. We tested the anticandidal activity of eugenol, main component of clove oil, and thymol, main component of thyme oil, alone or in combination, by investigating their ability to interfere with the architecture of the envelope of C. albicans. Both molecules alterated the morphogenesis of the envelope, but the effects of thymol were more pronounced than those of eugenol. Certain combinations of the two molecules led to a synergistic effect, which is interesting in the view of potentiating their inhibition of C. albicans colonisation and infectiousness.

Efficacy of an essential oil of Eugenia caryophyllata against Psoroptes cuniculi.

The acaricidal activity of Eugenia caryophyllata essential oil was evaluated in vitro and in vivo on Psoroptes cuniculi, a mange mite. In vitro, different concentrations of the oil were tested and the observed mites mortality was compared with that observed in untreated and treated (Acacerulen R) controls. In vivo, six P. cuniculi infected rabbits were topically treated with the oil diluted at 2.5% and compared with untreated and treated control groups of six rabbits each. In vitro, up to the concentration of 0.10% the oil gave highly significant (P<0.01) percentages of mite mortality respect to the untreated controls, but only up to 0.16% it showed the same efficacy of Acacerulen R. In vivo, the treatment with the essential oil cured all infested rabbits and no statistical differences were observed respect to the treated control group. The untreated rabbits remained infested.

Effects of eugenol on nerve and vascular dysfunction in streptozotocin-diabetic rats.

Hyperglycaemia in diabetes mellitus results in oxidative stress and pro-inflammatory changes which contribute to vascular complications including endothelial dysfunction and peripheral neuropathy. The aim of this study was to examine whether treatment with the dominant ingredient of clove oil, eugenol, which has antioxidant and anti-inflammatory properties, could improve diabetic vascular and nerve function in streptozotocin-induced diabetic rats. Intervention treatment was given for 2 weeks following 6 weeks of untreated diabetes. Dose-ranging studies on diabetic deficits in sciatic nerve motor and saphenous nerve sensory nerve conduction velocities gave ED50 values of 28 mg/kg and 9 mg/kg, respectively, conduction velocity being within the non-diabetic range at a dose of 200 mg/kg. Sciatic nerve endoneurial blood flow was 49% reduced by diabetes and this was completely corrected by 200 mg/kg eugenol treatment. Gastric fundus maximum nitrergic nerve-mediated relaxation was 44% reduced by diabetes; eugenol corrected this deficit by 69%. For renal artery rings, maximum endothelium-dependent relaxation to acetylcholine was 51% reduced by diabetes; eugenol corrected this deficit by 60%, with improvements in both nitric oxide and endothelium-derived hyperpolarising factor (EDHF)-mediated vasorelaxation components. Diabetes increased renal artery sensitivity to phenylephrine-mediated contraction, however, this was unaffected by eugenol treatment. Thus, aspects of both vascular and neural complications in experimental diabetes are improved by eugenol, which could have potential therapeutic implications for diabetic neuropathy and vasculopathy.

Antimicrobial activity of clove oil and its potential in the treatment of vaginal candidiasis.

In the present study, we evaluated antimicrobial activity of clove oil against a range of fungal pathogens including that responsible for urogenital infection. Clove oil was found to possess strong antifungal activity against opportunistic fungal pathogens such as Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, etc. The oil was found to be extremely successful in the treatment of experimental murine vaginitis in model animals. On evaluating various formulations, topical administration of the liposomized clove oil was found to be most effective against treatment of vaginal candidiasis.