Accelerated Wound Healing with a Diminutive Scar through Cocrystal Engineered Curcumin.

Pharmaceutical cocrystals ( Regulatory Classification of Pharmaceutical Co-Crystals Guidance for Industry; Food and Drug Administration, 2018) are crystalline solids produced through supramolecular chemistry to modulate the physicochemical properties of active pharmaceutical ingredients (APIs). Despite their extensive development in interdisciplinary sciences, this is a pioneering study on the efficacy of pharmaceutical cocrystals in wound healing and scar reducing. Curcumin-pyrogallol cocrystal (CUR-PYR) was accordingly cherry-picked since its superior physicochemical properties adequately compensate for limitative drawbacks of curcumin (CUR). CUR-PYR has been synthesized by a liquid-assisted grinding (LAG) method and characterized via FT-IR, DSC, and PXRD analyses. In vitro antibacterial study indicated that CUR-PYR cocrystal, CUR+PYR physical mixture (PM), and PYR are more effective against both Gram-negative (Pseudomonas aeruginosa and Escherichia coli) and Gram-positive (Staphylococcus aureus and Bacillus subtilis) bacteria in comparison with CUR. In vitro results also demonstrated that the viability of HDF and NIH-3T3 cells treated with CUR-PYR were improved more than those received CUR which is attributed to the effect of PYR in the form of cocrystal. The wound healing process has been monitored through a 15 day in vivo experiment on 75 male rats stratified into six groups: five groups treated by CUR-PYR+Vaseline (CUR-PYR.ung), CUR+PYR+Vaseline (CUR+PYR.ung), CUR+Vaseline (CUR.ung), PYR+Vaseline (PYR.ung), and Vaseline (VAS) ointments and a negative control group of 0.9% sodium chloride solution (NS). It was revealed that the wounds under CUR-PYR.ung treatment closed by day 12 postsurgery, while the wounds in other groups failed to reach the complete closure end point until the end of the experiment. Surprisingly, a diminutive scar (3.89 ± 0.97% of initial wound size) was observed in the CUR-PYR.ung treated wounds by day 15 after injury, followed by corresponding values for PYR.ung (12.08 ± 2.75%), CUR+PYR.ung (13.89 ± 5.02%), CUR.ung (16.24 ± 6.39%), VAS (18.97 ± 6.89%), and NS (20.33 ± 5.77%). Besides, investigating histopathological parameters including inflammation, granulation tissue, re-epithelialization, and collagen deposition signified outstandingly higher ability of CUR-PYR cocrystal in wound healing than either of its two constituents separately or their simple PM. It was concluded that desired solubility of the prepared cocrystal was essentially responsible for accelerating wound closure and promoting tissue regeneration which yielded minimal scarring. This prototype research suggests a promising application of pharmaceutical cocrystals for the purpose of wound healing.

A Fe3+-crosslinked pyrogallol-tethered gelatin adhesive hydrogel with antibacterial activity for wound healing.

In this work, a tunicate-inspired gelatin-based hydrogel is prepared by simply mixing 2,3,4-trihydroxybenzaldehyde (THB)-tethered gelatin solution with a small amount of Fe3+ ions via the Schiff-base reaction and simultaneous formation of hexavalent Fe-complexes. The resulting hydrogel (termed GelTHB-Fe) exhibits not only tunable gelation time, rheological properties and self-healing ability by adjusting the composition, but also robust adhesion to a variety of materials, with an average adhesion strength of 136.7 kPa, 147.3 kPa, 153.7 kPa, 92.9 kPa, and 56.5 kPa to PMMA, iron, ceramics, glass and pigskin, respectively. Intriguingly, the pyrogallol moieties impart an antibacterial activity to the GelTHB-Fe hydrogel, which is shown to reduce infection and promote wound healing in a diabetic rat model. This GelTHB-Fe hydrogel holds great potential as a promising tissue adhesive.

Metabolic Activation of Elemicin Leads to the Inhibition of Stearoyl-CoA Desaturase 1.

Elemicin is a constituent of natural aromatic phenylpropanoids present in many herbs and spices. However, its potential to cause toxicity remains unclear. To examine the potential toxicity and associated mechanism, elemicin was administered to mice for 3 weeks and serum metabolites were examined. Enlarged livers were observed in elemicin-treated mice, which were accompanied by lower ratios of unsaturated- and saturated-lysophosphatidylcholines in plasma, and inhibition of stearoyl-CoA desaturase 1 (Scd1) mRNA expression in liver. Administration of the unsaturated fatty acid oleic acid reduced the toxicity of 1'-hydroxylelemicin, the primary oxidative metabolite of elemicin, while treatment with the SCD1 inhibitor A939572 potentiated its toxicity. Furthermore, the in vitro use of recombinant human CYPs and chemical inhibition of CYPs in human liver microsomes revealed that CYP1A1 and CYP1A2 were the primary CYPs responsible for elemicin bioactivation. Notably, the CYP1A2 inhibitor α-naphthoflavone could attenuate the susceptibility of mice to elemicin-induced hepatomegaly. This study revealed that metabolic activation of elemicin leads to SCD1 inhibition in liver, suggesting that upregulation of SCD1 may serve as potential intervention strategy for elemicin-induced toxicity.

Pyrogallol induces the death of human pulmonary fibroblast cells through ROS increase and GSH depletion.

Pyrogallol (PG) inhibits the growth of various cells via stimulating O2•--mediated death. This study investigated the effects of PG on cell death in human pulmonary fibroblast (HPF) cells in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. PG inhibited the growth of HPF cells with an IC50 of ~50-100 µM at 24 h. PG induced a G1 phase arrest of the cell cycle and also triggered cell death accompanied by the loss of mitochondrial membrane potential (MMP; ∆ψm), Bcl-2 decrease, p53 increase and the activation of caspase-3. PG increased O2•- level in HPF cells and depleted GSH content in these cells. Z-VAD (a pan-caspase inhibitor) did not significantly change cell growth inhibition, death and MMP (∆ψm) loss in PG-treated HPF cells. N-acetylcysteine (NAC) attenuated growth inhibition, death and MMP (∆ψm) loss in PG-treated HPF cells and it decreased O2•- level in these cells as well. However, L-buthionine sulfoximine (BSO) strongly increased ROS level in PG-treated HPF cells and it intensified growth inhibition, cell death, MMP (∆ψm) loss and GSH depletion in these cells. In conclusion, PG-induced HPF cell death was closely related to increases in ROS level and GSH depletion.

Quantitation of tr-cinnamaldehyde, safrole and myristicin in cola-flavoured soft drinks to improve the assessment of their dietary exposure.

Quantitation of tr-cinnamaldehyde, safrole and myristicin was carried out in 70 samples of cola-flavoured soft drinks purchased in eight European countries with the purpose of assessing the variability in the levels of these substances. Results indicated a limited variability in the content of the three substances: the ratio between the 90th and the 10th percentile concentration amounted to 21, 6 and 13 for tr-cinnamaldehyde, safrole and myristicin, respectively. The uncertainty in the assessment of dietary exposure to these substances due to the variability of their level in cola-flavoured drinks was low. Based on these analytical data and on refined food consumption data, estimates of exposure to safrole associated to cola drink consumption, along with Margin of Exposure (MOE) values, were obtained. For high consumers of cola-flavoured soft drinks in certain age groups, within some European countries, MOE values lower than 10,000 resulted, MOE values of 10,000 or higher having been stated by the EFSA as a quantitative criterion to identify low concern from a public health point of view and low priority for risk management actions. The lowest MOE values, from 1900 to 3000, were observed for children and teen agers in the United Kingdom and Ireland.

Toxicology and carcinogenesis studies of pyrogallol (CAS No. 87-66-1) in F344/N rats and B6C3F1/N mice (dermal studies).

UNLABELLED: The current main commercial use of pyrogallol is the production of pharmaceuticals and pesticides. In analytical chemistry, pyrogallol is used as a complexing agent, reducing agent, and, in alkaline solution, as an indicator of gaseous oxygen. Pyrogallol was nominated for testing by private individuals based on its frequent occurrence in natural and manufactured products, including hair dyes, and the apparent lack of carcinogenicity data. Male and female F344/N rats and B6C3F1/N mice were administered pyrogallol (99% pure) dermally for 3 months or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, Escherichia coli, mouse bone marrow cells, and mouse peripheral blood erythrocytes. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats received dermal applications of pyrogallol in 95% ethanol at doses of 0, 9.5, 18.75, 37.5, 75, or 150 mg pyrogallol/kg body weight, 5 days per week for 14 weeks. Additional groups of 10 male and 10 female special study rats were administered the same doses, 5 days per week for 23 days. All rats survived until the end of the study except for one vehicle control female. Mean body weights of dosed groups of males and females were generally similar to those of the vehicle controls. Chemical-related clinical findings included brown staining and irritation of the skin at the site of application. There were no changes in the hematology, serum clinical chemistry, thyroid hormone values, or organ weights attributable to the dermal administration of pyrogallol. The incidences of squamous hyperplasia, hyperkeratosis, and chronic active inflammation of the skin at the site of application were significantly increased in all dosed groups of males and females. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice received dermal applications of pyrogallol in 95% ethanol at doses of 0, 38, 75, 150, 300, or 600 mg pyrogallol/kg body weight, 5 days per week for 14 weeks. All mice survived until the end of the study. Mean body weights of dosed groups of males and females were similar to those of the vehicle controls. Chemical-related clinical findings included brown staining and irritation at the site of application. There were no changes in the hematology values or organ weights attributable to the dermal administration of pyrogallol. The incidences of squamous hyperplasia, hyperkeratosis, and chronic active inflammation of the skin at the site of application were significantly increased in all dosed groups of males and females. The incidence of hematopoietic cell proliferation of the spleen in 600 mg/kg males was significantly greater than that in the vehicle control group. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats received dermal applications of pyrogallol in 95% ethanol at doses of 0, 5, 20, or 75 mg pyrogallol/kg body weight, 5 days per week for up to 104 weeks. Survival of dosed groups of male and female rats was similar to that of the vehicle control groups. Mean body weights of dosed male and female rats were similar to those of the vehicle control groups throughout the study. Irritation of the skin at the site of application was the only chemical-related clinical finding and occurred in the 20 and 75 mg/kg groups. In the skin at the site of application, there were significant increases in the incidences of hyperplasia in all dosed groups of males and females, hyperkeratosis in 20 and 75 mg/kg males and all dosed groups of females, inflammation in 75 mg/kg males and 20 and 75 mg/kg females, and sebaceous gland hyperplasia in 20 and 75 mg/kg males and females. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice received dermal applications of pyrogallol in 95% ethanol at doses of 0, 5, 20, or 75 mg pyrogallol/kg body weight, 5 days per week for up to 105 weeks. Survival of dosed groups of male mice was similar to that of the vehicle control group. Survival was significantly decreased in 75 mg/kg females; most early deaths in this group were due to ulcers at or adjacent to the site of application. The mean body weights of 75 mg/kg female mice were generally over 10% less than those of the vehicle controls during year 2 of the study. Irritation and/or ulceration of the skin at the site of application were the only chemical-related clinical findings and occurred predominantly in the 20 and 75 mg/kg groups. In the skin at the site of application, the incidence of squamous cell carcinoma in 75 mg/kg females was significantly greater than that in the vehicle control group. Two 75 mg/kg males had squamous cell papillomas; squamous cell papillomas have not been observed in historical control male mice in four ethanol dermal studies. Increased incidences of nonneoplastic lesions at the site of application included hyperplasia and hyperkeratosis in all dosed groups; inflammation, fibrosis, and pigmentation in the 20 and 75 mg/kg groups; and sebaceous gland hyperplasia and ulcer in the 75 mg/kg groups. Similar lesions in the skin of the neck and back immediately adjacent to the site of application were observed; the incidences of hyperplasia, hyperkeratosis, ulcer, inflammation, and fibrosis at these sites were significantly increased in 75 mg/kg male and female mice, and the incidence of sebaceous gland hyperplasia was significantly increased in 75 mg/kg female mice. Dermal application of pyrogallol also resulted in significant increases in the incidences of bone marrow hyperplasia in males and females and lymphoid hyperplasia of the axillary, inguinal, and mandibular lymph nodes; adrenal cortical hematopoietic cell proliferation; and mammary gland hyperplasia in females. GENETIC TOXICOLOGY: Pyrogallol was tested in two independent bacterial mutation studies; both studies gave positive results in one or more strains of S. typhimurium or E. coli. In the first study, positive results were seen in S. typhimurium strain TA100 with and without S9 exogenous metabolic activation, and negative results were obtained in strain TA98. In the second study, which was conducted with the same lot of pyrogallol that was used in the 3-month and 2-year studies, positive results were obtained in S. typhimurium strains TA98, TA100, and in E. coli strain WP2 uvrA/pKM101 in the absence of S9. With S9, this sample of pyrogallol was mutagenic in the E. coli strain but gave equivocal responses in S. typhimurium strains TA98 and TA100. In vivo, a micronucleus test that measured frequency of micronucleated polychromatic erythrocytes in bone marrow of male B6C3F1/N mice following three intraperitoneal injections of pyrogallol, gave negative results. In a second in vivo test, no increase in the frequency of micronucleated erythrocytes was observed in the peripheral blood of female B6C3F1/N mice treated with pyrogallol via dermal application for 3 months; in male mice, however, results were equivocal, based on a significant increase in micronucleated erythrocytes observed at a single dose level at the end of the 3-month study. CONCLUSIONS: Under the conditions of these 2-year dermal studies, there was no evidence of carcinogenic activity of pyrogallol in male or female F344/N rats administered 5, 20, or 75 mg/kg. There was equivocal evidence of carcinogenic activity of pyrogallol in male B6C3F1/N mice based on increased incidences of squamous cell papilloma of the skin at the site of application. There was some evidence of carcinogenic activity of pyrogallol in female B6C3F1/N mice based on increased incidences of squamous cell carcinoma of the skin at the site of application. Dermal administration of pyrogallol caused increased incidences of nonneoplastic lesions of the skin at the site of application in male and female rats and mice, skin adjacent to the site of application in male and female mice, and mammary gland in female mice.

Genotoxic and apoptotic activities of the food flavourings myristicin and eugenol in AA8 and XRCC1 deficient EM9 cells.

Some food flavourings, such as safrole and methyleugenol, are known for their genotoxic and hepatocarcinogenic properties whereas for others, such as myristicin, there is less data. Myristicin and eugenol are both alkenylbenzenes, and we compared their direct genotoxicity in repair proficient (AA8) and repair deficient XRCC(-) (EM9) Chinese hamster ovary cells. Cell viability was assessed by the MTT assay. The comet assay was used to evaluate DNA breaks, and the γ-H2AX assay to evaluate induction of double strand breaks. We assessed apoptosis by measuring caspases activation, and the TUNEL assay. Reduction of cell viability was similar in AA8 and EM9 cells, for both compounds. After 1h eugenol produced DNA strand breaks in the comet assay and induced double strand breaks in the γ-H2AX assay in AA8 cells, while myristicin was not genotoxic in both the comet and the γ-H2AX assays. Both flavourings were negative in EM9 cells. After 24h eugenol and myristicin induced DNA fragmentation detected by TUNEL in both cell lines, but only myristicin activated caspases. Myristicin was more apoptotic than eugenol, in both cell lines. The XRCC1 protein does not influence the apoptotic activity of either compound.

Hepatoprotective effect of myristicin from nutmeg (Myristica fragrans) on lipopolysaccharide/d-galactosamine-induced liver injury.

To evaluate the hepatoprotective activity of spices, 21 different spices were fed to rats with liver damage caused by lipopolysaccharide (LPS) plus d-galactosamine (D-GalN). As assessed by plasma aminotranferase activities, nutmeg showed the most potent hepatoprotective activity. Bioassay-guided isolation of the active compound from nutmeg was carried out in mice by a single oral administration of the respective fractions. Myristicin, one of the major essential oils of nutmeg, was found to possess extraordinarily potent hepatoprotective activity. Myristicin markedly suppressed LPS/D-GalN-induced enhancement of serum TNF-alpha concentrations and hepatic DNA fragmentation in mice. These findings suggest that the hepatoprotective activity of myristicin might be, at least in part, due to the inhibition of TNF-alpha release from macrophages. However, further studies are needed to elucidate the hepatoprotective mechanism(s) of myristicin.

Induction of emesis in Suncus murinus by pyrogallol, a generator of free radicals.

1. We investigated whether or not pyrogallol, a generator of free radicals, is emetogenic in Suncus murinus, the house musk shrew. Pyrogallol (i.p.) caused dose-dependent emesis in suncus with an ED50 value of 77.3 mg kg-1. At a dose of 128 mg kg-1, all suncus vomited with mean latency of 18.8 +/- 5.2 min and the number of vomiting episodes was 8.6 +/- 2.9. 2. The prophylactic effects of N-(2-mercaptopropionyl)-glycine (MPG), an antioxidant, and tropisetron, a 5-hydroxytryptamine3 (5-HT3) receptor antagonist, were studied. Pyrogallol (128 mg kg-1, i.p.)-induced emesis was prevented by treatment with MPG (i.p.) or tropisetron (s.c.) with ID50 values of 149 mg kg-1 and 117 micrograms kg-1, respectively. 3. Pyrogallol-induced emesis was completely prevented by surgical abdominal vagotomy. 4. The present results indicate that pyrogallol-induced emesis is characteristically very similar to that caused by cisplatin and support the idea that generation of free radicals causes the release of peripheral 5-HT, which stimulates vagal afferent sensory nerves to cause emesis.

The influence of topical dermatological treatment modalities on epidermal Langerhans cells and contact sensitization in mice.

The influence of the widely used topical dermatological treatment modalities anthralin, coal tar and pyrogallol on surface markers of epidermal Langerhans cells and contact sensitization was studied and compared with that of a PUVA treatment. A common effect of all dermatological therapies tested was inhibition of Langerhans cell ATPase, whereas an effect on MHC class II antigens was found only after PUVA or tar treatment. The induction of contact hypersensitivity was inhibited only by PUVA, and not by the other treatments. These results show that various forms of topical therapy influence surface markers and immunological function of epidermal Langerhans cells differently.

Acute toxicity testing in the nonlethal dose range: a new approach.

A new modification of acute toxicity testing of chemicals in rats is presented. Instead of using death as the principal criterion of toxicity, it is based on a careful, standardized clinical assessment of toxic signs measured in the nonlethal dose range. Test compounds are administered to groups of rats at four dose levels, selected on the basis of pilot experiments. General indices of toxicity, i.e., body weight gain, food and water consumption, and body temperature, are recorded at regular intervals; activity in the home cage is monitored continuously with a newly developed passive infrared device; neurobehavioral dysfunctions are assessed repeatedly with a checklist; and routine hematology is done on the 4th day after administration of the test compounds. All measured signs of toxicity are scored in relation to the control groups so that the absolute magnitude of the score increases as a function of the deviation from the normal conditions of the animals. In order to take into consideration the course of intoxication and the rate of recovery, toxicity scores are added over the duration of the whole experiment. For each variable measured, the resulting total scores are converted into a relative point system, scaling from 0 to 10 points. These points are added to a single "total ToxScore" value for each dose group. A regression line is calculated for these total ToxScore points, and the dose giving 15 total ToxScore points is determined. Based on the results obtained with nine reference substances, a classification system is proposed that is comparable to that based on LD50 values. It is concluded that the proposed test procedure provides much more information on the signs of acute toxicity, the course of the intoxication, the slope of the dose-effect curve, and the rate of recovery than does the standard LD50 test. In addition, the degree of distress and suffering of the animals is reduced.

Pyrogallol in the tumour stage of mycosis fungoides: a case report.

The case history of a patient with mycosis fungoides (tumour stage) is reported. As ultimum refugium pyrogallol 5% in petrolatum proved to be remarkably effective.