Multi-targeted effects of D-carvone against Non-Small Cell Lung Cancer (NSCLC): A network pharmacology-based study.

Non-small cell lung cancer (NSCLC) is a complex malignancy with a high degree of heterogeneity, representing approximately 85% of all lung cancer cases. The treatment landscape for NSCLC has been revolutionised by incorporating targeted and immunotherapies; however, novel therapeutic modalities are consistently needed to enhance the treatment outcomes. Indeed, alternative anti-cancer therapies involving natural products have drawn the attention of clinicians and scientists owing to their remarkable chemopreventive potential, often displaying minimal toxicity. D-carvone (CN) is one such natural product that has exhibited numerous promising therapeutic benefits, yet its efficacy against NSCLC remains enigmatic. In the present study, network pharmacological studies and molecular docking in conjunction with in-vitro validation were used to elucidate the underlying mechanism of action of CN comprehensively. Different databases revealed a total of 77 putative anti-NSCLC targets of CN. The identified core targets were utilised to construct a "Compound- Target- Disease" network by Cytoscape (v3.9.0). Further analysis identified 5 core/ hub targets of CN including JAK2, ERK1, ESR1, GSK3B and HSP90AA1. Molecular docking indicated a strong binding interaction of the compound with these core targets. Also, Gene Ontology and KEGG analysis validated the involvement of multiple biological processes. Additionally, CN significantly inhibited cell proliferation, clonogenicity, and wound healing potential while promoting apoptosis in a dose-dependent manner in H1299 and A549 cell lines as examined by flow cytometry, morphological assessment, and western blotting. In conclusion, this study delineates the therapeutic effects of CN on NSCLC, thus highlighting CN as a putative drug candidate for further analysis.

Chemical Composition and Phytotoxic Activity of Mentha vagans Boriss. Essential Oil.

This study explored the composition of essential oil (EO) and the first phytotoxic screening of EO obtained from the stems and leaves of Mentha vagans Boriss (MVEO) via hydro-distillation technique. The EO ingredients were detected through Gas Chromatography-Mass Spectrometry (GC-MS). GC-MS analysis revealed that MVEO contained 49 constituents, constituting 93.95 % of the total oil. Among MVEO constituents, dihydrocarvone was observed as the dominant constituent (24.14%), followed by D-carvone (16.28%) and piperitone (18.14%). The phytotoxic effects of MVEO and its dominant compounds were examined against Amaranthus retroflexus, Lolium perenne, and Poa annua. Significant inhibition was observed by MVEO in comparison with the major constituents and their mixture, suppressing the seedling growth of tested species at the lowest dosage (0.01 mg/mL); in general, seedling growth of all tested species was markedly inhibited when applied concentration of the EO and its constituents reached 0.05 mg/mL. Our results also indicated that constituents other than the dominant compounds of MVEO possessed considerable phytotoxic effects because the EO's activity was stronger than its major constituents and their mixture. Thus, additional studies are required to investigate MVEO and its constituents and commercialize them as environment-friendly bio-herbicides.

d-Carvone inhibits the JAK/STAT3 signaling pathway and induced the apoptotic cell death in the human gastric cancer AGS cells.

Globally, gastric cancer is one of the leading cause of death. Surgical and chemotherapy constitute an important treatment regimen. Unfortunately, less than 20 persons out of 100 patients are live on almost 5 years. Hence, a nontoxic, effective and significantly enhancing novel therapeutic agent is required. d-Carvone is a natural terpenoid present in the essential oils and abundant in the seeds of caraway, as well as known folk medication for diarrhea, acidity, and other gastric disorders. Nevertheless, the role of d-carvone on gastric cancer and its underlying molecular mechanism resides enigmatic. Cells were treated with d-carvone to find out the IC50 by MTT assay. This study shows that 20 and 25 µM d-carvone has induced the reactive oxygen species production and mitochondrial membrane potential in gastric cancer AGS cells, which were evaluated by 2,7-dichlorofluoresceindiacetate and Rh123 staining methods, respectively. The effect of d-carvone against the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway was studied through immunoblotting. Then, we found that it effectively inhibited the proliferation of cell, and the induction of cell apoptosis was scrutinized by dual, 4',6-diamidino-2-phenylindole, and also propidium iodide staining methods. We also explored the fundamental molecular signaling mechanism of the d-carvone and our data depicts that d-carvone induced apoptosis cell death by mitochondrial reactive oxygen species production and downregulation of the and JAK and STAT3 signaling molecules. These overall findings support that the d-carvone inhibits the JAK/STAT3 signaling pathway and induces cell death in the gastric cancer AGS cells.

Anti-arthritic activity of D-carvone against complete Freund's adjuvant-induced arthritis in rats through modulation of inflammatory cytokines.

Chronic joint pain due to loss of cartilage function, degradation of subchondral bone, and related conditions are common plights of an arthritis patient. Antioxidant compounds could solve the problems in arthritic condition. The objective of this study was to evaluate the anti-arthritic activity of D-carvone against complete Freund's adjuvant (CFA)-induced arthritis in rats. D-carvone was orally administered for 25 days at the doses of 30 and 60 mg/kg against CFA-induced arthritic rats. Changes in body weight, paw swelling, organ index, hematological parameters, oxidative stress markers, inflammatory cytokines, and histopathology were recorded. Oral treatment of D-carvone significantly improved the body weight, reduced the paw swelling, edema formation, and organ index in arthritic rats. The levels of white blood cells were reduced, red blood cells and hemoglobin levels were improved in D-carvone treated arthritic rats. Lipid peroxidation levels were lowered whereas enzymatic and non-enzymatic antioxidants were significantly elevated by D-carvone administration against arthritic rats. D-carvone significantly modulated inflammatory cytokine levels and improved the ankle joint pathology against CFA-induced arthritic inflammation. In conclusion, D-carvone proved significant anti-arthritic activity against CFA-induced arthritis in rats.

Safety and efficacy of a feed additive consisting of an essential oil obtained from the fruit of Carum carvi L. (caraway oil) for all animal species (FEFANA asbl).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of an essential oil obtained from the fruit of Carum carvi L. (caraway oil), when used as a sensory additive in feed and water for drinking for all animal species. The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) Panel concluded that the use of caraway oil is of no concern up to the following concentrations in complete feed: 9 mg/kg for chickens for fattening, 13 mg/kg for laying hens, 12 mg/kg for turkeys for fattening, 16 mg/kg for piglets, 19 mg/kg for pigs for fattening, 24 mg/kg for sows, 35 mg/kg for veal calves (milk replacer), 11 mg/kg for cattle for fattening, 10 mg/kg for dairy cows, sheep, goats, horses and rabbits, 25 mg/kg for salmonids and dogs. These conclusions were extrapolated to other physiologically related species. For cats, ornamental fish and other species, no conclusion can be drawn. The use of caraway oil in animal feed under the proposed conditions of use is safe for the consumer and the environment. The additive under assessment should be considered as an irritant to skin and eyes, and as a respiratory and skin sensitiser. When handling the essential oil, exposure of unprotected users to perillaldehyde may occur. Therefore, to reduce the risk, the exposure of the users should be minimised. Since C. carvi and its preparations were recognised to flavour food and its function in feed would be essentially the same as that in food, no further demonstration of efficacy was considered necessary.

D-Carvone Attenuates CCl4-Induced Liver Fibrosis in Rats by Inhibiting Oxidative Stress and TGF-ß 1/SMAD3 Signaling Pathway.

D-carvone is a natural monoterpene found in abundance in the essential oil of aromatic medicinal plants with a wide range of pharmacological values. However, the impact of D-carvone on liver fibrosis remains unclear. This study aimed to evaluate the anti-fibrotic potential of D-carvone in a rat model of liver fibrosis and to clarify the possible underlying mechanisms. Liver fibrosis was induced in rats by carbon tetrachloride, CCl4 (2.5 mL/kg, interperitoneally every 72 h for 8 weeks). Oral treatment of rats with D-carvone (50 mg/kg, daily) started on the 3rd week of CCl4 administration. D-carvone significantly enhanced liver functions (ALT, AST), oxidant/antioxidant status (MDA, SOD, GSH, total antioxidant capacity; TAC), as well as histopathological changes. Moreover, D-carvone effectively attenuated the progression of liver fibrosis, evident by the decreased collagen deposition and fibrosis score by Masson trichrome staining (MT) and α-SMA protein expression. Moreover, D-carvone administration resulted in a significant downregulation of the pro-fibrogenic markers TGF-ß1 and SMAD3 and upregulation of MMP9. These findings reveal the anti-fibrotic effect of D-carvone and suggest regulation of the TGF-ß1/SMAD3 pathway, together with the antioxidant activity as a mechanistic cassette, underlines this effect. Therefore, D-carvone could be a viable candidate for inhibiting liver fibrosis and other oxidative stress-related hepatic diseases. Clinical studies to support our hypothesis are warranted.

D-carvone induced ROS mediated apoptotic cell death in human leukemic cell lines (Molt-4).

The immature lymphoid cells with chromosomal structural and numerical abnormalities cause the acute lymphoblastic leukemia (ALL). This hematologic disorder constitutes about 25% of cancer prognosis among children and adolescents. D-Carvone, a monocyclic monoterpene obtained from the essential oils extracted from plants is reported to possess the various biological activities. The present study was aimed to investigate the anticancer potential of D-Carvone against the human leukemic Molt-4 cells. The cytotoxicity of DCarvone was analyzed by MTT assay. The level of lipid peroxidation and antioxidants were determined. The intracellular ROS, MMP and apoptosis were demonstrated by fluorescent staining techniques. The MTT assay revealed that the D-Carvone treatment suppressed the viability of Molt-4 cells and the IC50 was determined at 20 µM/ml. The D-Carvone treatment was increased the oxidative stress and reduced the level of antioxidants in the Molt-4 cell lines. The increased intracellular ROS, apoptotic cell death, and diminished MMP was noted in the D-Carvone treatment. In the Molt-4 cells, D-carvone induced the apoptosis in a time and dose dependent manner by the activation of caspases-8, -9 and -3. Thus, data provide insights for the clinical application of D-Carvone in the treatment of blood cancer Molt-4 cells. Our study suggests the therapeutic potential D-Carvone for the treatment of leukemia in future.

Ougan juice debittering using ultrasound-aided enzymatic hydrolysis: Impacts on aroma and taste.

The optimal sonication conditions (40 kHz, 80 W/L and 60 min) during Ougan juice debittering by Aspergillus niger koji extract were established. Enzymatic hydrolysis degrees of naringin and limonin were enhanced to 89.90% and 36.16%, and enzymatic hydrolysis time was shortened by 33%. Sonication significantly enhanced activities of α-l-rhamnosidases, ß-glucosidases and limoninases from A. niger koji extract and facilitated break of CO bonds in naringin (p < 0.05). These accounted for the enhanced enzymatic hydrolysis degrees and velocities of bitter compounds. Meanwhile, sonication lowered 40%, 7% and 21%, 13%, 11%, 25% of bitter, sour tastes and green, citrus-like, floral, woody notes, but enhanced 18% and 15% of fruity and sweet notes, resulting in 38% and 33% increases in over-all taste and aroma scores. Lowered levels of bitter compounds, organic acids, green, citrus-like, floral, woody aroma compounds and enhanced levels of fruity, sweet aroma compounds caused by sonication accounted for the flavor improvements.

D-Carvone inhibit cerebral ischemia/reperfusion induced inflammatory response TLR4/NLRP3 signaling pathway.

To explore the present treatment strategies for ischemic stroke lowered by ischemia-reperfusion (I/R) injury, to hypothesize the effect of d-Carvone on cerebral I/R brain injury induced neuroinflammation through oxidative stress markers mechanism via NRLP3 and TLR4 marker expressions in rat model. The rats were divided into four groups: Sham, I/R vehicle, I/R + D-carvone (10 mg/kg/bw), I/R + D-carvone (20 mg/kg/bw). Supplementation of d-carvone at dose of 10 and 20 m/kg/bw increased the water content, reduced infract volume, attenuated neurological score depicts, furthermore it had antioxidative, anti-inflammatory, and anti-apoptotic effects against cerebral I/R brain injury. In the brain tissues decreased proinflammatory cytokines IL-1ß and TNF-α reduced interleukins IL-6, IL-4, IL-10 & VEGF dose dependently, and mRNA expressions of NLRP3, caspase -1, TNF-α, ASC, IL-1ß and TLR3 down regulated in cerebral I/R induced rats. Finally d- carvone can successfully improve the cerebral I/R induced rats neuroinflammation, in the hippocampus and cortical areas of the brain finally reduces cerebral I/R induced injury. These results were hypothesized that d-carvone contributed to cerebral stroke associated with the TLR3, giving an excellent therapeutic approach for cerebral I/R brain injury.

Gas chromatography-mass spectrometry combined with multivariate data analysis as a tool for differentiating between processed orange juice samples on the basis of their volatile markers.

The contents of volatile aroma compounds in orange juice vary after high hydrostatic pressure (HHP) processing and pasteurization, and hence gas chromatography-mass spectrometry has potential for discriminating between orange juice samples according to the processing method. Effects of HHP processing and pasteurization on orange juice were investigated immediately after processing and after storage for 28 days at 4 °C. Processing decreased the contents of most terpenes and esters and increased those of alcohols and aldehydes. Volatile compounds that could serve as discriminant indicators of HHP processing and pasteurization were heptanal, (E)-2-heptenal, (E)-2-nonenal, and d-carvone and ß-terpineol, p-mentha-1,5-dien-8-ol, carveol, and ß-copaene, respectively. The discrimination abilities of pH, ascorbic acid, total soluble solids, and color were compared with those of volatile compounds, which were found to be lower. The compounds d-carvone and ß-terpineol could be used as discriminant indicators of HHP-treated and pasteurized orange juice, respectively, throughout the storage period.

Preventive effect of D-carvone during DMBA induced mouse skin tumorigenesis by modulating xenobiotic metabolism and induction of apoptotic events.

The structural integrity and excellent immune system of the skin makes it a protective covering, inspite of its exposure to hazardous compounds. In the present study, the chemopreventive efficacy of D-carvone was studied in 7, 12-dimethylbenz[a]anthracene (DMBA) induced skin carcinogenesis. DMBA (25 µg in 0.1 m L-1acetone) was used to induce skin cancer in Swiss albino mice. Animals were randomly divided into six groups of six animals in each. Different concentrations of D-carvone (10, 20, 30 mg/kg body weight) were used to assess its anticancer effect. Tumor incidence, tumor volume, tumor burden, histological examination and levels of phase I and phase II detoxification agents were analyzed in experimental animals. Further, expression of p53 and various apoptotic proteins including- Bcl-2, Bax was analyzed using immunohistochemistry and enzymatic expression of apoptotic proteins caspase-3 and caspase-9 was carried out by using ELISA. We observed 100% tumor incidence in DMBA-painted animals and our results showed that D-carvone at 20 mg dose significantly prevents skin carcinogenesis. Our results also showed decreased levels of phase I enzymes (Cyt P450 and-Cyt b5) with increased levels of phase II enzymes (GR, GST and GSH) and increased expression of Bax, caspase-3 and caspase-9 with decreased expression of mutated p53 and Bcl-2 in animals treated with DMBA and D-carvone at 20 mg dose. The results of the present study suggest that D-carvone can be used as a chemopreventive agent against skin cancer, as it induces apoptosis in cancer. However, further studies are warranted to check chemopreventive efficacy of D-carvone on cell proliferation, angiogenesis, and metastasis before going to human trial.

Peer review of the pesticide risk assessment of the active substance carvone (substance evaluated d-carvone).

The conclusions of the EFSA following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State, the Netherlands and co-rapporteur Member State, Sweden, for the pesticide active substance carvone are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012. The conclusions were reached on the basis of the evaluation of the representative use of carvone (substance evaluated d-carvone) as a plant growth regulator on seed potatoes. The reliable end points, appropriate for use in regulatory risk assessment, are presented. Missing information identified as being required by the regulatory framework is listed.