Isoeugenol Research Articles

Comparative analysis of phenolic profile of Monodora myristica and Monodora tenuifolia

This study sought to identify phenolics present in the seeds of Monodora myristica and Monodora tenuifolia by Gas chromatography (GC) coupled to Flame ionization detector (FID). GC-FID analysis identified fifty-three different types of phenolics in both M. myristica and M. tenuifolia seeds. Predominant phenolics are Myristicin (RT: 13.78), Caffeic acid (RT: 14.16), Safrole (RT: 11.37), Methyl Eugenol (RT: 12.32), Catechin (RT: 3.63), Elemicin (RT: 13.69), Quercetin (RT: 25.05), Kaempherol (RT: 21.52), Methyl Isoeugenol (RT: 12.69) and Eugenol (RT: 11.70). It was observed that M. myristica is rich in Myristicin (42.60%), Caffeic acid (23.39%), Elemicin (3.82%) and Eugenol (1.02%), while M. tenuifolia is rich in Safrole (11.86%), Methyl Eugenol (6.28%), Catechin (5.27%), Quercetin (2.97%), Kaempherol (2.27%) and Methyl Isoeugenol (1.45%). However, the two species of Monodora contained virtually the same kinds of phenolics but in varying quantities with M. myristica having a higher total phenolic content (1478.32 mg/100 g) than M. tenuifolia (1171.52 mg/100 g). Both species are promising sources of phenolics. Key words: Monodora myristica, Monodora tenuifolia, phenolics.

Toxicokinetics of Isoeugenol in F344 rats and B6C3F1 mice

1. Isoeugenol (IEG) has been tested for toxicity and carcinogenicity due to high potential for human exposure and the structural resemblance to known carcinogenic allylbenzenes. In order to support the interpretation of toxicity and carcinogenecity study outcomes, a toxicokinetic study was performed in which both sexes of F344 rats and B6C3F1 mice were given IEG as a single intravenous (IV) or gavage administration.2. Following IV administration, IEG was rapidly eliminated from systemic circulation in both species and sexes. Gavage administration revealed a rapid absorption of IEG with tmax values ≤20 min for both species and sexes. In rats, AUC increased in a greater than dose-proportional manner and Clapp values decreased with increasing dose in both sexes suggesting saturation of IEG metabolism. On the other hand, Clapp values in male mice increased with increasing dose suggesting induction of IEG metabolism although this was not evident in the females.3. Absolute bioavailability was greater in female rats (19%) than male rats (10%) (p < 0.0001), but was not different between the sexes for mice (28% males; 31% females) (p = 0.2437). The collective toxicokinetic data supported that low bioavailability following administration of IEG was the result of extensive first-pass metabolism.

Neuroprotective Efficacy of Eugenol and Isoeugenol in Acrylamide-Induced Neuropathy in rats: Behavioral and Biochemical evidence

The primary objective of this investigation was to assess the neuroprotective efficacy of spice active principles namely Eugenol (Eug) and isoeugenol (IE) in an acrylamide (ACR) neuropathy model in rats. In the present study, ACR administration (50 mg/kg bw, i.p. 3 times/week) for 5 weeks to growing rats caused typical symptoms of neuropathy. We found that treatment of ACR rats with spice active principles (10 mg/kg bw, for 5 weeks) caused marked improvement in gait score and responses in a battery of behavioral tests. Terminally, both spice active principles markedly attenuated ACR-induced markers of oxidative stress viz., reactive oxygen species (ROS), malondialdehyde (MDA) and nitric oxide (NO) in sciatic nerve (SN) as well as brain regions (cortex Ct, cerebellum Cb). Treatment with Eug restored the reduced glutathione levels in SN and brain regions. Interestingly, both spice active principles effectively diminished ACR-induced elevation in cytosolic calcium levels and acetylcholinesterase activity in SN and Ct. Further, the diminished activity of ATPase among ACR rats was enhanced in SN and restored in brain regions. Furthermore, Eug treatment significantly offset ACR-induced depletion in dopamine levels in brain regions. Collectively our findings suggest the propensity of these spice active principles to attenuate ACR-induced neuropathy. Further studies are necessary to understand the precise molecular mechanism/s by which these spice active principles attenuate neuropathy. Nevertheless, our data clearly demonstrate the beneficial effects of spice active principles in ACR-induced neuropathy in rats and suggest their possible therapeutic usage as an adjuvant in the management of other forms of neuropathy in humans.

Evidence of acrylamide induced oxidative stress and neurotoxicity in Drosophila melanogaster – Its amelioration with spice active enrichment: Relevance to neuropathy

Acrylamide (ACR) intoxication in its monomeric form leads to neuronal damage in both experimental animals and humans. Oxidative stress is one of the principle mechanisms related to the neurotoxicity of ACR exposure. Hence, the present study aimed to recapitulate the potential of ACR to cause oxidative stress and neurotoxic effects in Drosophila melanogaster. Exposure of adult male flies (Oregon K strain) to ACR (1–10mM, 7 d) in the diet resulted in a concentration and time dependent mortality, while the survivors exhibited significant locomotor deficits. Further, ACR exposure (1–5mM, 3 d) caused robust oxidative stress as evidenced by markedly elevated levels of reactive oxygen species and hypdroperoxides in head/body regions. Enhanced lipid peroxidation, perturbations in the activities of antioxidant enzymes accompanied with depletion of reduced glutathione levels in head region at high concentrations suggested induction of oxidative stress. Further, marked diminution in the activities of complexes I–III, Succinic dehydrogenase, with concomitant reduction in MTT suggested the propensity of ACR to impair mitochondrial function. Furthermore, ACR-induced neurotoxic effects were discernible in terms of diminished ATPase activity, enhanced activity of acetylcholinesterase and dopamine depletion. In a satellite study, employing a co-exposure paradigm, we tested the propensity of spice actives namely eugenol (EU) and isoeugenol (IE) to ameliorate ACR-induced neurotoxicity. EU/IE enriched diet offered marked protection against ACR-induced mortality, locomotor dysfunctions and oxidative stress. Furthermore, the spice actives prevented the depletion of reduced GSH levels, maintained the activity of AChE enzyme and dopamine levels in head region. Collectively, these findings clearly demonstrate that ACR induced neurotoxicity in Drosophila may be mediated through oxidative stress mechanisms and the potential of spice actives to abrogate the condition. These data suggest that Drosophila may serve as a suitable model to understand the possible mechanism/s associated with ACR associated neuropathy.

Soda/AQ Pulping of a Hardwood with Trapping of the Generated Coniferyl Alcohol

Coniferyl alcohol is generated from the cleavage of β-O-4 bonds during alkaline pulping of hardwoods and softwoods. Once formed, some of the coniferyl alcohol (CA) is transformed into vinylguaiacol (VG) and isoeugenol (IE). All three of these compounds (CA, VG, and IE) can rearrange to quinone methides (QMs) under alkaline conditions and become involved in condensation reactions with lignin and carbohydrate moieties. The aim of this investigation was to see if ethylguaiacol (EG) addition to soda-AQ (SAQ) pulping systems would lead to its reaction with the QMs from CA, VG, and IE to form dimers that are unreactive towards further condensation. Sugar maple wood meal was delignified by the SAQ process with EG added at 5.0 wt% on wood meal. Three dimers were detected in the pulping effluent and identified as a combination of EG with CA, VG, and IE. The total yield of the three dimers corresponded to ∼80% of the estimated CA that would have been generated. Conventional kraft, SAQ, and SAQ+EG pulping of the maple chips were then performed. The bleachability of the SAQ+EG pulp was significantly improved as compared to the SAQ pulp and it was estimated to be superior to the kraft pulp as well.

Bioproduction of vanillin using an organic solvent-tolerant Brevibacillus agri 13

Nowadays, majority of vanillin supplied to the world market is chemically synthesized from a petroleum-based raw material, raising a concern among the consumers regarding the product safety. In this study, an organic solvent-tolerant Brevibacillus agri 13 previously reported for a strong predilectic property was utilized as a whole-cell biocatalyst for bioproduction of vanillin from isoeugenol (IG). B. agri 13 is the first biocatalyst reported for bioproduction of vanillin at a temperature as high as 45°C. Both pH and temperature were found to affect vanillin production significantly. An extreme level of organic solvent tolerance of B. agri 13 allowed us to utilize it in a biphasic system using organic solvents generally considered as highly toxic to most bacteria. With an addition of butyl acetate at 30% (v/v) as an organic second phase, toxicity of IG exerted onto the biocatalyst was reduced dramatically while faster and more efficient vanillin production was obtained (1.7 g/L after 48 h with 27.8% molar conversion).

Use of the human monocytic leukemia THP-1 cell line and co-incubation with microsomes to identify and differentiate hapten and prohapten sensitizers

Consumer and medical products can contain leachable chemical allergens which can cause skin sensitization. Recent efforts have been directed at the development of non-animal based tests such as in vitro cell activation assays for the identification of skin sensitizers. Prohapten identification by in vitro assays is still problematic due to the lack of prohapten bioactivation. The present study evaluated the effect of hapten and prohapten exposure on cell surface markers expression (CD86, CD54 and CD40) in the human monocytic leukemia, THP-1, cell line. Upregulation of activation and costimulatory markers are key events in the allergic sensitization process and have been reported to serve as indicators of skin sensitization. Cells were exposed to the prohaptens benzo(a)pyrene (BaP), 7,12-dimethylbenz(a)anthracene (DMBA), carvone oxime (COx), cinnamic alcohol (CA) and isoeugenol (IEG) at concentrations ranging from 1 to 10 μM for 24 and 48 h. The direct-binding haptens dinitrochlorobenzene (DNCB), benzoquinone (BQ), hydroxylethyl acrylate (HEA) and benzylbromide (BB) were used as positive controls. Cells were also exposed to the irritants sodium dodecyl sulfate (SDS) and sulfanilamide (SFA). Bioactivation of prohaptens was achieved by adding aroclor-induced rat liver microsomes (S9) to the cell cultures. Consistent upregulation of surface expressions of CD86, CD54 (ICAM-1) and CD40 was observed in THP-1 cells treated with direct-acting haptens (±S9) or prohapten (+S9). Upregulation of these markers was not observed after exposure to skin irritants or prohaptens in the absence of exogenously added S9. In conclusion, modification of in vitro cell culture assays to include co-incubation with microsomes enhances identification of prohaptens and allows them to be clearly distinguished from direct-binding haptens.

Identification of PDL-1 as a novel biomarker of sensitizer exposure in dendritic-like cells

The development of novel in vitro methods to assess risks of allergic sensitization are essential in reducing animal testing whilst maintaining consumer safety. The main research objectives of this study were to identify novel biomarkers to assess the sensitization predictability of chemicals. Phenotypic and cytokine responses of moDCs and MUTZ-3 cells were investigated following application of contact sensitizers; dinitrochlorobenzene (DNCB), cinnamaldehyde (Cin), eugenol (E), isoeugenol (IE), P-phenylenediamine (PPD) and non-sensitizers; salicyclic acid (SA) and sodium lauryl sulphate (SLS). CD86 was up-regulated on MUTZ-3 cells in response to DNCB, Cin and PPD, however, moDCs only modulated CD86 in response to DNCB and E. PDL-1 (Programmed death receptor ligand-1) proved a promising sensitization biomarker in MUTZ-3 cells where up-regulation occurred in response to DNCB, Cin, IE and PPD. Additionally, moDC-expressed PDL-1 was modulated in response to Cin, IE and E thus demonstrating improved sensitizer predictability when compared with CD86. MCP-1 and RANTES were identified as biomarkers of DNCB exposure but MCP-1 did not show any change in expression above controls for the other sensitizers investigated. However, RANTES was increased in MUTZ-3 cells by both DNCB and Cin. Our findings highlight novel biomarkers which, in MUTZ-3 cells, could be taken forward within a multiple biomarker in vitro assay ensuring strong and reliable predictability.

Experimental inhalation of fragrance allergens in predisposed subjects: effects on skin and airways

Exposure to fragrances is increasingly encountered in the environment. Some fragrances are known to be important skin and potential airway sensitizers. We investigated whether patients with contact allergy to isoeugenol (ISO) or hydroxyisohexyl-3-carboxaldehyde (HICC) would react to inhalation exposure at the level of the airways and skin. Eleven patients sensitized to ISO and 10 patients sensitized to HICC were exposed for 60 min to 1000 microg m(-3) of these compounds in an exposure chamber at rest, and to geraniol 1000 microg m(-3) as a control. Patients wore protective clothing to prevent skin exposure. Assessments were performed prior to exposure, and immediately, 2, 5, 24 and 72 h afterwards. There were no significant changes in lung function but a tendency towards an increased bronchial hyper-responsiveness after exposure to any of the compounds. Laboratory parameters of inflammation did not indicate responses. Single patients reported respiratory symptoms unrelated to objective measures. In contrast, the observed skin symptoms corresponded to the patients' specific sensitization. Four patients reported symptoms compatible with delayed-type hypersensitivity, and two demonstrated a flare after ISO. On re-exposure they did not respond to a lower, more realistic level of ISO. Inhalation of high concentrations of fragrance contact allergens apparently poses a risk for some patients of developing manifest haematogenic contact dermatitis, while the changes in the respiratory tract are limited to symptoms in some subjects without objective changes.

TRPA1 receptor localisation in the human peripheral nervous system and functional studies in cultured human and rat sensory neurons

TRPA1 is a receptor expressed by sensory neurons, that is activated by low temperature (<17 degrees C) and plant derivatives such as cinnamaldehyde and isoeugenol, to elicit sensations including pain. Using immunohistochemistry, we have, for the first time, localised TRPA1 in human DRG neurons, spinal cord motoneurones and nerve roots, peripheral nerves, intestinal myenteric plexus neurones, and skin basal keratinocytes. TRPA1 co-localised with a subset of hDRG neurons positive for TRPV1, the heat and capsaicin receptor. The number of small/medium TRPA1 positive neurons (< or =50 microm) was increased after hDRG avulsion injury [percentage of cells, median (range): controls 16.5 (7-23); injured 46 (34-55); P<0.005], but the number of large TRPA1 neurons was unchanged [control 19.5 (13-31); injured 21 (11-35)]. Similar TRPA1 changes were observed in cultured hDRG neurons, after exposure to a combination of key neurotrophic factors NGF, GDNF and NT-3 (NTFs) in vitro. We used calcium imaging to examine responses of HEK cells transfected with hTRPA1 cDNA, and of human and rat DRG neurons cultured with or without added NTFs, to cinnamaldehyde (CA) and isoeugenol (IE). Exposure to NTFs in vitro sensitized cultured human sensory neuronal responses to CA; repeated CA exposure produced desensitisation. In rDRG neurons, low (225 microM) CA preincubation enhanced capsaicin responses, while high (450 microM and 2mM) CA caused inhibition which was partially reversed in the presence of 8 bromo cAMP, indicating receptor dephosphorylation. While TRPA1 localisation is more widespread than TRPV1, it represents a promising novel drug target for the treatment of chronic pain and hypersensitivity.

Advantage of using CBA/N strain mice in a non-radioisotopic modification of the local lymph node assay

The murine local lymph node assay (LLNA) is currently recognized as a stand-alone test method for determining the skin sensitizing potential of chemicals. It has been incorporated into the official test guidelines published by some authorities, including the OECD. To avoid the use of radioisotopes, efforts have been made recently to develop non-radioisotopic modifications of the LLNA. A non-radioisotopic modification of the LLNA was developed previously using 5-bromo-2'-deoxyuridine (BrdU) incorporation (non-RI LLNA). However, the non-RI LLNA was found to be somewhat less sensitive than the standard assay. This study reports the advantage of using mice of the CBA/N strain in the non-RI LLNA to improve the sensitivity of this method. The non-RI LLNA was performed using CBA/JN and CBA/N mice exposed to one of four confirmed skin sensitizers, 2,4-dinitrochlorobenzene (DNCB), eugenol (EG), isoeugenol (IEG) or alpha-hexylcinnamic aldehyde (HCA), and to one non-sensitizer, propylene glycol (PG). The EC3 values for DNCB, IEG, EG, HCA and PG were calculated to be 0.1%, 9.6%, 40.6%, 45.5% and >50% in CBA/JN mice and 0.08%, 1.9%, 10.7%, 20.3% and >50% in CBA/N mice, respectively. The EC3 values for DNCB, IEG, EG, HCA and PG in the standard LLNA using CBA/Ca mice and radioisotopes were reported elsewhere as being 0.08%, 1.3%, 13.0%, 8.0% and >50%, respectively. The EC3 values derived from the CBA/N mice in the non-RI LLNA were nearly equivalent to the EC3 values obtained using the standard radioisotopic LLNA with CBA/Ca mice. These data suggest that the use of CBA/N mice may provide a realistic opportunity to develop a version of the LLNA that does not have a requirement for the use of radioisotopes, but which nevertheless has sensitivity approaching, or comparable to, the standard method.

Effects of Visible Light Irradiation on Eugenol-treated Oral Mucosa

The purpose of this study was to evaluate the histopathological effects of eugenol (EUG) and iso-eugenol (IsoEUG)--with or without visible light (VL) irradiation--on oral mucous membranes. Oral mucous membranes of mice were applied with three agents, EUG, IsoEUG, and aceton (as the control) in the absence or presence of VL irradiation. VL irradiation resulted in more tissue damage for EUG- or IsoEUG-treated mucosa compared to corresponding compounds without VL irradiation, and that damage under IsoEUG treatment was greater than that under EUG treatment. Necrosis, but not apoptosis, was preferentially expressed in EUG- or IsoEUG-treated mucous membranes in the presence of VL irradiation.

Abnormal solvent effects on hydrogen atom abstraction. 2. Resolution of the curcumin antioxidant controversy. The role of sequential proton loss electron transfer.

The rates of reaction of 1,1-diphenyl-2-picrylhydrazyl (dpph*) radicals with curcumin (CU, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), dehydrozingerone (DHZ, "half-curcumin"), and isoeugenol (IE) have been measured in methanol and ethanol and in two non-hydroxylic solvents, dioxane and ethyl acetate, which have about the same hydrogen-bond-accepting abilities as the alcohols. The reactions of all three substrates are orders of magnitude faster in the alcohols, but these high rates can be suppressed to values essentially equal to those in the two non-hydroxylic solvents by the addition of acetic acid. The fast reactions in alcohols are attributed to the reaction of dpph* with the CU, DHZ, and IE anions (see J. Org. Chem. 2003, 68, 3433), a process which we herein name sequential proton loss electron transfer (SPLET). The most acidic group in CU is the central keto-enol moiety. Following CU's ionization to a monoanion, ET from the [-(O)CCHC(O)-](-) moiety to dpph* yields the neutral [-(O)CCHC(O)-]* radical moiety which will be strongly electron withdrawing. Consequently, a phenolic proton is quickly lost into the alcohol solvent. The phenoxide anion so formed undergoes charge migration to produce a neutral phenoxyl radical and the keto-enol anion, i.e., the same product as would be formed by a hydrogen atom transfer (HAT) from the phenolic group of the CU monoanion. The SPLET process cannot occur in a nonionizing solvent. The controversy as to whether the central keto-enol moiety or the peripheral phenolic hydroxyl groups of CU are involved in its radical trapping (antioxidant) activity is therefore resolved. In ionizing solvents, electron-deficient radicals will react with CU by a rapid SPLET process but in nonionizing solvents, or in the presence of acid, they will react by a slower HAT process involving one of the phenolic hydroxyl groups.

In vitro and in vivo effects of phenolic antioxidants against cisplatin-induced nephrotoxicity.

We have investigated the effect of phenolic antioxidants on cisplatin-induced cytotoxicity in vero (African Green Monkey Kidney) cells and in rat renal cortical slices in vitro, and on cisplatin-induced nephrotoxicity in rats in vivo. Incubation of cisplatin with vero cells resulted in time- and concentration-dependent cytotoxicity, as characterized by decreased tryphan blue exclusion (TBE) and increased release of lactate dehydrogenase (LDH) into the medium. Cisplatin also caused reduction of glutathione (GSH) in a concentration-dependent manner. In the rat renal cortical slices model, incubation of cisplatin for 120 min caused an increase in malondialdehyde (MDA), a decrease in GSH and inhibited p-aminohippurate (PAH) uptake in a concentration-dependent manner. Among phenolic antioxidants, isoeugenol (IG) was found to be more active against cisplatin-induced cytotoxicity in vero cells as well as in rat renal cortical slices than eugenol (EG) and dehydrozingerone (DZ). However none of the test compounds were able to arrest the reduction of the GSH content induced by cisplatin in either the vero cells or the renal cortical slice model. Administration of cisplatin (3 mg/kg) i.p. to rats resulted in significant reduction of body weight, and elevation of blood urea nitrogen (BUN) and serum creatinine. Treatment with IG 10 mg/kg i.p. 1 h before cisplatin resulted in partial but significant protection against the cisplatin-induced reduction of body weight, and elevation of BUN and serum creatinine, the protection being 34, 46, and 62%, respectively. EG and DZ (10 mg/kg, i.p.) were found to be inactive in vivo. Because IG is a potent free radical scavenger and protects against cisplatin-induced toxicitiy, the present results have many clinical implications in chemotherapy and thus warrants further investigation.

Physico-Chemical Properties and Antioxidant Activities of Methoxy Phenols

A few structurally related phenols, dehydrozingerone (DZ), bromopentenone (BP), eugenol (EG) and isoeugenol (IEG), derived from plant products show antioxidant properties by inhibiting lipid peroxidation in membrane models. The phenoxyl radicals produced by the scavenging of free radicals during the inhibition of lipid peroxidation, were also generated by specific one-electron oxidants using pulse radiolysis. The radical lifetimes (second order rate constants for radical-radical reactions), reactivities with hydroxyl and model peroxyl radicals and the one-electron reduction potentials with respect to the standard couples were quantified. These results along with their lipophilicity data were correlated with their antioxidant activity (IC 50 values).

The noninvasive mouse ear swelling assay: II. Testing the contact sensitizing potency of fragrances

The noninvasive mouse ear swelling assay (MESA) for contact allergy testing was evaluated using fragrance components and complex fragrance mixtures. The test materials represented weak sensitizers and nonsensitizers. Two versions of the MESA were investigated. Both were noninvasive and utilized only topical abdominal dosing and ear challenge with single applications in BALB cBy mice. The vit A MESA differed from the regular MESA only in that mice were maintained on a diet with 17-fold higher levels of vitamin A (vit A) acetate beginning 3 weeks prior to induction. Sensitization reactions were determined by measuring the mean increase in ear swelling over baseline at 24, 48 and 72 hr postexposure. Irritation doseresponse curves facilitated choosing a high nonirritating challenge dose. Sensitization dose-response curves were developed for cinnamaldehyde (CINN) and a complex fragrance mixture, F-16. From these curves, the SD50 was determined. This value represents the dose which sensitized half the animals and serves to rank the potency of compounds for allergic contact dermatitis and to compare values among different assays. The SD50 for CINN was 21.6% while the SD50 vit A for F-16 was 26.6%. The other fragrance, isoeugenol (ISOE), and fragrance mixtures, F-07 and F-22, were also found to be weak sensitizers in the MESA and vit A MESA. The results in the MESA for CINN and ISOE were in the range observed with guinea pig test protocols but showed that the MESA was more sensitive than human test protocols. Two of the fragrance mixtures tested in the MESA gave comparable results in the Buehler guinea pig assay. However, the third (F-22) was negative in the Buehler assay and the MESA, but positive in the vit A MESA. The results of this work with weak sensitizers and the companion study (Thorne et al., 1991) with potent sensitizers at low doses illustrate that the noninvasive MESA is as sensitive as many standard guinea pig assays. In addition, it is easier and much less expensive to perform. The vit A MESA has the sensitivity and predictive power needed to test compounds and mixtures for contact sensitizing potency.

Factors influencing conjugation of phenolic compounds with the epsilon-amino group.

ABSTRACTFactors influencing the conjugation potency of eugenol (E), isoeugenol (IE), methyl isoeugenol (MIE), and acetyl isoeugenol (AIE) with [l‐14C]ɛ‐aminocaproic acid are investigated. The conjugation reaction for IE is accelerated in the presence of H2O2‐peroxidase and inhibited under anaerobic conditions by nitrogen gas flow. An oxidation reaction is found to be necessary for the conjugation of IE with ɛ‐aminocaproic acid. Immediately after mixing the phenolic compounds with [1‐14C]ɛ‐aminocaproic acid and H2O2‐peroxidase, the main conjugates are 7.7% for IE, 2.1% for E, 0.7% for AIE, and 0.2% for MIE. IE, which is a potent sensitizer in vivo, produces the greatest amount of conjugates while MIE, which is negative in vivo, produces almost no conjugates. AIE and E, both mild sensitizers in vivo, produce less conjugates than IE.