Molecular interactions between metformin and D-limonene inhibit proliferation and promote apoptosis in breast and liver cancer cells.

BACKGROUND: Cancer is a fatal disease that severely affects humans. Designing new anticancer strategies and understanding the mechanism of action of anticancer agents is imperative. HYPOTHESIS/PURPOSE: In this study, we evaluated the utility of metformin and D-limonene, alone or in combination, as potential anticancer therapeutics using the human liver and breast cancer cell lines HepG2 and MCF-7. STUDY DESIGN: An integrated systems pharmacology approach is presented for illustrating the molecular interactions between metformin and D-limonene. METHODS: We applied a systems-based analysis to introduce a drug-target-pathway network that clarifies different mechanisms of treatment. The combination treatment of metformin and D-limonene induced apoptosis in both cell lines compared with single drug treatments, as indicated by flow cytometric and gene expression analysis. RESULTS: The mRNA expression of Bax and P53 genes were significantly upregulated while Bcl-2, iNOS, and Cox-2 were significantly downregulated in all treatment groups compared with normal cells. The percentages of late apoptotic HepG2 and MCF-7 cells were higher in all treatment groups, particularly in the combination treatment group. Calculations for the combination index (CI) revealed a synergistic effect between both drugs for HepG2 cells (CI = 0.14) and MCF-7 cells (CI = 0.22). CONCLUSION: Our data show that metformin, D-limonene, and their combinations exerted significant antitumor effects on the cancer cell lines by inducing apoptosis and modulating the expression of apoptotic genes.

Bioactivities and Synergistic Effect of Elsholtzia ciliata Essential Oil and Its Main Components against Lasioderma serricorne.

Investigations have shown that storage bugs seriously harm grains during storage. In the interim, essential oils (EOs) have been proven to be a good botanical pesticide. The anti-Lasioderma serricorne properties of Elsholtzia ciliata essential oil, which was obtained by steam distillation, were evaluated using DL-limonene, carvone, and their two optical isomer components using contact, repelling, and fumigation techniques. Simultaneously, the fumigation, contact, and repellent activities of carvone and its two optical isomers mixed with DL-limonene against L. serruricorne were evaluated. The results showed that E. ciliata, its main components (R-carvone, DL-limonene), and S-carvone exhibited both fumigations (LC50 = 14.47, 4.42, 20.9 and 3.78 mg/L) and contact (LD50 = 7.31, 4.03, 28.62 and 5.63 µg/adult) activity against L.serricorne. A binary mixture (1:1) of R-carvone and DL-limonene displayed an obvious synergistic effect. A binary mixture (1:1) of carvone and its two optical isomers exhibited an obvious synergistic effect, too. Furthermore, the repellent activity of the EO, carvone, and its two optical isomers, DL-limonene, and a combination of them varied. To stop insect damage during storage, E. ciliata and its components can be utilized as bio-insecticides.

Synergistic interactions of essential oil components with antibiotics against multidrug-resistant Corynebacterium striatum.

AIM: In this study, it was aimed to examine the antibacterial activity of the essential oil components (EOCs), carvacrol (CAR), cinnamaldehyde (CIN), thymol (TH), alpha pinene (α-PN), eucalyptol (EU), limonene (LIM), and the antibiotics, linezolid (LZD), vancomycin (VAN), gentamicin (GEN), ciprofloxacin (CIP), clindamycin (CLN), and penicillin (PEN) against 50 multidrug resistant Corynebacterium striatum strains, and the synergistic interactions of CAR and CIN with the antibiotics against 10 randomly selected Coryne. striatum strains to explore synergistic interactions to determine if their combined use could enhance antibiotic activity and potentially reduce resistance. METHODS AND RESULTS: The activity of the EOCs and the antibiotics against Coryne. striatum strains isolated from clinical specimens, was examined by broth microdilution method. The synergistic interactions of the EOCs with the antibiotics against 10 randomly selected Coryne. striatum strains were determined by checkerboard method. EOCs, CIN, and CAR and antibiotics, LZD, VAN, GEN, CIP, and CLN were detected to have antibacterial activity against Coryne. striatum strains alone and either synergistic interactions were observed in combinations of the antibiotics with EOCs. CONCLUSIONS: All Coryne. striatum strains were determined to be susceptible to VAN and LZD and resistant to GEN, PEN, CIP, and CLN. Synergistic interactions were observed in all combinations of antibiotics tested with CAR and CIN.

Assessment of the in vitro acaricidal activity of Bravecto® (fluralaner) and a proposed orange oil-based formulation vehicle for the treatment of Sarcoptes scabiei.

BACKGROUND: Sarcoptic mange is a serious animal welfare concern in bare-nosed wombats (Vombatus ursinus). Fluralaner (Bravecto®) is a novel acaricide that has recently been utilised for treating mange in wombats. The topical 'spot-on' formulation of fluralaner can limit treatment delivery options in situ, but dilution to a volume for 'pour-on' delivery is one practicable solution. This study investigated the in vitro acaricidal activity of Bravecto, a proposed essential oil-based diluent (Orange Power®), and two of its active constituents, limonene and citral, against Sarcoptes scabiei. METHODS: Sarcoptes scabiei were sourced from experimentally infested pigs. In vitro assays were performed to determine the lethal concentration (LC50) and survival time of the mites when exposed to varying concentrations of the test solutions. RESULTS: All compounds were highly effective at killing mites in vitro. The LC50 values of Bravecto, Orange Power, limonene and citral at 1 h were 14.61 mg/ml, 4.50%, 26.53% and 0.76%, respectively. The median survival times of mites exposed to undiluted Bravecto, Orange Power and their combination were 15, 5 and 10 min, respectively. A pilot survival assay of mites collected from a mange-affected wombat showed survival times of < 10 min when exposed to Bravecto and Orange Power and 20 min when exposed to moxidectin. CONCLUSIONS: These results confirm the acaricidal properties of Bravecto, demonstrate acaricidal properties of Orange Power and support the potential suitability of Orange Power and its active constituents as a diluent for Bravecto. As well as killing mites via direct exposure, Orange Power could potentially enhance the topical delivery of Bravecto to wombats by increasing drug penetration in hyperkeratotic crusts. Further research evaluating the physiochemical properties and modes of action of Orange Power and its constituents as a formulation vehicle would be of value.

D-limonene inhibits peritoneal adhesion formation in rats via anti-inflammatory, anti-angiogenic, and antioxidative effects.

The aim of this research was to investigate the effects of D-limonene on decreasing post-operative adhesion in rats and to understand the mechanisms involved. Peritoneal adhesions were induced by creating different incisions and excising a 1 × 1 cm section of the peritoneum. The experimental groups included a sham group, a control group in which peritoneal adhesions were induced without any treatment, and two treatment groups in which animals received D-limonene with dosages of 25 and 50 mg/kg after inducing peritoneal adhesions. Macroscopic examination of adhesions showed that both treatment groups had reduced adhesion bands in comparison to the control group. Immunohistochemical assessment of TGF-ß1, TNF-α, and VEGF on day 14 revealed a significant increment in the level of immunopositive cells for the mentioned markers in the control group, whereas administration of limonene in both doses significantly reduced levels of TGF-ß1, TNF-α, and VEGF (P < 0.05). Induction of peritoneal adhesions in the control group significantly increased TGF-ß1, TNF-α, and VEGF on days 3 and 14 in western blot evaluation, while treatment with limonene significantly reduced TNF-α level on day 14 (P < 0.05). Moreover, VEGF levels in both treatment groups significantly reduced on days 3 and 14. In the control group, a significant increment in the levels of MDA and NO and a notable decline in the levels of GPX, CAT was observed (P < 0.05). Limonene 50 group significantly reduced MDA level and increased GPx and CAT levels on day 14 (P < 0.05). In summary, D-limonene reduced adhesion bands, inflammatory cytokines, angiogenesis, and oxidative stress.

Essential oil from Citrus depressa peel exhibits antimicrobial, antioxidant and cancer chemopreventive effects.

BACKGROUND: Many diseases may be caused by pathogens and oxidative stress resulting from carcinogens. Earlier studies have highlighted the antimicrobial and antioxidant effects of plant essential oils (EO). It is crucial to effectively utilize agricultural waste to achieve a sustainable agricultural economy and protect the environment. The present study aimed to evaluate the potential benefits of EO extracted from the discarded peels of Citrus depressa Hayata (CD) and Citrus microcarpa Bunge (CM), synonyms of Citrus deliciosa Ten and Citrus japonica Thunb, respectively. RESULTS: Gas chromatography-mass spectrometry analysis revealed that the main compounds in CD-EO were (R)-(+)-limonene (38.97%), γ-terpinene (24.39%) and linalool (6.22%), whereas, in CM-EO, the main compounds were (R)-(+)-limonene (48.00%), ß-pinene (13.60%) and γ-terpinene (12.07%). CD-EO exhibited inhibitory effects on the growth of common microorganisms, including Candida albicans, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. However, CM-EO showed only inhibitory effects on E. coli. Furthermore, CD-EO exhibited superior antioxidant potential, as demonstrated by its ability to eliminate 1,1-diphenyl-2-picrylhydrazyl and 2,2'-azinobis-3-ethylbenzthiazoline-6-sulfonate free radicals. Furthermore, CD-EO at a concentration of 100 µg mL-1 significantly inhibited 12-O-tetradecanoylphorbol-13-acetate-induced cancer transformation in mouse epidermal JB6 P+ cells (P < 0.05), possibly by up-regulating protein expression of nuclear factor erythroid 2-related factor 2 and its downstream antioxidant enzymes, such as NAD(P)H:quinone oxidoreductase 1, heme oxygenase-1 and UGT1A. CONCLUSION: These findings suggest that CD-EO exhibits inhibitory effects on pathogenic microorganisms, possesses antioxidant properties and has cancer chemopreventive potential. © 2024 Society of Chemical Industry.

In vitro antiproliferative and apoptotic effects of thiosemicarbazones based on (-)-camphene and R-(+)-limonene in human melanoma cells.

A series of 38 thiosemicarbazone derivatives based on camphene and limonene were evaluated for their antiproliferative activity. Among them, 19 were synthesized and characterized using proton and carbon-13 nuclear magnetic resonance (1H and 13C NMR). For initial compound selection, human melanoma cells (SK-MEL-37) were exposed to a single concentration of a compound (100 µM) for 24, 48, and 72 hours, and cell detachment was visually observed. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Nineteen compounds (4, 6, 8, 11, 13, 14, 15, 16, 17, 18, 20, 22, 25, 26, 31, 3', 4', 6', and 9') yielded cell viability below 20%. Subsequently, IC50 values for these compounds were determined, ranging from 11.56 to 55.38 µM, after 72 hours of treatment. Compound 17 (o-hydroxybenzaldehyde (-)-camphene-based thiosemicarbazone) demonstrated the lowest IC50 value, followed by compound 4 (benzaldehyde (-) camphene-based thiosemicarbazone) at 12.84 µM. Regarding compound 4, we observed the induction of a characteristic ladder pattern of DNA fragmentation through gel electrophoresis. Furthermore, fluorescence, flow cytometry and scanning microscopy assays revealed morphological changes consistent with apoptosis induction. Additionally, the measurement of caspase 6 and 8 activity in cellular extracts after treatment for 2, 4, 6, and 24 hours suggested the potential involvement of the extrinsic apoptosis pathway in the mechanism of action of compound 4. Further investigations, including molecular docking studies, are required to fully explore the potential of compound 4 and the other selected compounds, highlighting their promising role in future melanoma therapy research.

Effect of vitamin E, D-limonene, and their combination on nulliparous rabbit reproductive performance.

Oxidative status is important in reproductive performance and using two natural antioxidants is more beneficial than one in nulliparous rabbits. The goal, effect of vitamin E (VitE), D-limonene (DL), and VitE+DL on maternal LBW (MLBW), conception (CR), pregnancy (PR), and kindling rates (KR), gestation length (GL), total litter size at birth (TLSB) and weaning (TLSW), live kits at birth (LKB) and weaning (LKW), dead kits at birth (DKB) and weaning (DKW), kits mortality rate at weaning (KMRW), Kit weight at birth (KWB) and weaning (KWW), total kit weight at birth (TKWB) and weaning (TKWW), and concentrations of progesterone (P4) and Malondialdehyde (MDA), during first two pregnancies. A total of 24 healthy female WNZ rabbits were randomly selected and assigned into four groups (6/each). Control (animals injected with 1.0 mL propylene glycol), VitE (60 mg IM injection/animal, 2X/week pre-mating and 3X post-mating until mid-pregnancy, DL (20 mg IM injection/animal, 2X/week pre-mating and 1X at mating, and VitE+DL (IM injection/animal with the same doses and times applied in VitE and DL groups. All animals were treated during 1st pregnancy only. The results confirmed that animals treated with VitE+DL gained significant maternal LBW in 1st pregnancy, reduced dead kit number at birth and kit mortality rate at weaning, increased live kits and total kit weight at birth and weaning in the two pregnancies, and also increased significantly kit weight at birth and weaning in the treatments than controls in the two pregnancies, and DL was greater in 1st pregnancy. Progesterone concentrations in mid-pregnancy rose significantly in VitE+DL during 1st pregnancy and DL in 2nd pregnancy. Malondialdehyde concentrations dropped significantly in VitE and VitE+DL in mid-pregnancy in the two pregnancies. Eventually, the integration of VitE and DL displayed their unique properties for improving productive and reproductive performance in nulliparous rabbits.

Limonene anti-TMV activity and its mode of action.

The main component of orange peel essential oil is limonene. Limonene is a natural active monoterpene with multiple functions, such as antibacterial, antiseptic and antitumor activity, and has important development value in agriculture. This study found that limonene exhibited excellent anti-tobacco mosaic virus (TMV) bioactivity, with results showing that its protection activity, inactivation activity, and curative activity at 800 µg/mL were 84.93%, 59.28%, and 58.89%, respectively-significantly higher than those of chito-oligosaccharides. A direct effect of limonene on TMV particles was not observed, but limonene triggered the hypersensitive response (HR) in tobacco. Further determination of the induction activity of limonene against TMV demonstrated that it displayed good induction activity at 800 µg/mL, with a value of 60.59%. The results of physiological and biochemical experiments showed that at different treatment days, 800 µg/mL limonene induced the enhancement of defense enzymes activity in tobacco, including of SOD, CAT, POD, and PAL, which respectively increased by 3.2, 4.67, 4.12, and 2.33 times compared with the control (POD and SOD activities reached highest on the seventh day, and PAL and CAT activities reached highest on the fifth day). Limonene also enhanced the relative expression levels of pathogenesis related (PR) genes, including NPR1, PR1, and PR5, which were upregulated 3.84-fold, 1.86-fold and 1.71-fold, respectively. Limonene induced the accumulation of salicylic acid (SA), and increased the relative expression levels of genes related to SA biosynthesis (PAL) and reactive oxygen species (ROS) burst (RBOHB), which respectively increased by 2.76 times and 4.23 times higher than the control. Systemic acquired resistance (SAR) is an important plant immune defense against pathogen infection. The observed accumulation of SA, the enhancement of defense enzymes activity and the high-level expression of defense-related genes suggested that limonene may induce resistance to TMV in tobacco by activating SAR mediated by the SA signaling pathway. Furthermore, the experimental results demonstrated that the expression level of the chlorophyll biosynthesis gene POR1 was increased 1.72-fold compared to the control in tobacco treated with 800 µg/mL limonene, indicating that limonene treatment may increase chlorophyll content in tobacco. The results of pot experiment showed that 800 µg/mL limonene induced plant resistance against Sclerotinia sclerotiorum (33.33%), Phytophthora capsici (54.55%), Botrytis cinerea (50.00%). The bioassay results indicated that limonene provided broad-spectrum and long-lasting resistance to pathogen infection. Therefore, limonene has good development and utilization value, and is expected to be developed into a new botanical-derived anti-virus agent and plant immunity activator in addition to insecticides and fungicides.

In Silico Prediction of Selected Bioactive Compounds Present in Alpinia elegans (C.Presl) K.Schum Seed Oil as Potential Drug Candidates Against Human Cancer Cell Lines.

OBJECTIVE: Alpinia elegans (Zingiberaceae) is a Philippine endemic plant reported to have various folkloric uses. The seed oil of A. elegans has been shown to contain a majority of the following bioactive compounds: D-limonene, α-pinene, and caryophyllene oxide. The study sought to determine if the bioactive compounds found in A. elegans seed oil would be a good natural, inexpensive, and less-detrimental alternative for cancer treatment. METHODS: The study utilized in silico (Way2Drug predictive services, SwissADME, AutoDock 4) experiment to examine the aforementioned compounds as viable therapeutic candidates against human cancer cell lines. RESULT: Results determined that the compounds D-limonene, α-pinene, and caryophyllene oxide were most potent against thyroid gland carcinoma (8505C) cells, brain glaucoma (Hs 683) cells, and promyeloblast leukemia (HL-60) cells, respectively. Additionally, D-limonene was the only compound to show arrhythmia as an adverse effect. Predictions showed that the compounds could inhibit cellular growth factors and serine/threonine-protein kinase activity. The compounds generated a bioavailability score of 0.55 and exhibited blood-brain barrier (BBB) penetration. D-limonene, α-pinene, and caryophyllene oxide had binding energy of -4.59, -5.43, and -6.92, respectively. CONCLUSION: The binding energy indicated that the ligands could securely dock to the receptors, thus suggesting that interaction between the ligands and receptors was stable. Results have shown that the compounds are promising candidates against human cancer cell lines by inhibiting cell proliferation and inducing apoptosis.

Antiproliferative Activity and Ultrastructural Changes in Promastigote and Amastigote forms of Leishmania amazonensis Caused by Limonene-Acylthiosemicarbazide Hybrids.

Leishmaniasis is a tropical zoonotic disease. It is found in 98 countries, with an estimated 1.3 million people being affected annually. During the life cycle, the Leishmania parasite alternates between promastigote and amastigote forms. The first line treatment for leishmaniasis are the pentavalent antimonials, such as N-methylglucamine antimoniate (Glucantime®) and sodium stibogluconate (Pentostam®). These drugs are commonly related to be associated with dangerous side effects such as cardiotoxicity, nephrotoxicity, hepatotoxicity, and pancreatitis. Considering these aspects, this work aimed to obtain a new series of limonene-acylthiosemicarbazides hybrids as an alternative for the treatment of leishmaniasis. For this, promastigotes, axenic amastigotes, and intracellular amastigotes of Leishmania amazonensis were used in the antiproliferative assay; J774-A1 macrophages for the cytotoxicity assay; and electron microscopy techniques were performed to analyze the morphology and ultrastructure of parasites. ATZ-S-04 compound showed the best result in both tests. Its IC50 , in promastigotes, axenic amastigotes and intracellular amastigotes was 0.35±0.08 µM, 0.49±0.06 µM, and 15.90±2.88 µM, respectively. Cytotoxicity assay determined a CC50 of 16.10±1.76 µM for the same compound. By electron microscopy, it was observed that ATZ-S-04 affected mainly the Golgi complex, in addition to morphological changes in promastigote forms of L. amazonensis.

Combination of tamoxifen and D-limonene enhances therapeutic efficacy in breast cancer cells.

Breast cancer one of the most common diseases in women, has a high death and morbidity rate. Tamoxifen being very much effective in the chemoprevention of breast cancer has been shown to develop resistance during the course of treatment making it difficult for patient's survival. By combining tamoxifen with naturally occurring substances having similar activities, might control the toxicity and increase the susceptibility towards the treatment. As a natural compound, D-limonene has been reported to inhibit the growth of certain malignancies significantly. The main goal of our work is to investigate the combinatorial antitumor effects of D-limonene and tamoxifen in MCF-7 cells, as well as understand the potential underlying anticancer mechanism. MTT assays, colony formation assays, DAPI and Annexin V-FITC labeling, flow cytometer analysis, and western blot analysis were used to explore the details of anticancer mechanism. The combined effects of tamoxifen with D-limonene have shown significant decrease in the cell viability of MCF 7 cells. According to flow cytometer analyses and Annexin V/PI staining, D-limonene has been found to increase tamoxifen-mediated apoptosis as compared to the treatment alone in these cells. Additionally, cell growth has been found to be arrested at G1 phase by regulating cyclin D1 and cyclin B1. Our research consequently provided the first evidence that combining D-limonene and tamoxifen might increase the anticancer efficacy by inducing apoptosis in MCF 7 breast cancer cells. This combinatorial treatment strategy demands more research which might fulfill the need for improved treatment efficacy in controlling breast cancer.

L-limonene reduces aortic artery atherosclerosis by inhibiting oxidative stress/inflammatory responses in diabetic rats fed high-fat diet.

Atherosclerosis, a leading cause of mortality worldwide, is driven by multiple risk factors such as diabetes. Oxidative stress and inflammation assist interrelated roles in diabetes-accelerated atherosclerosis. Thereby, treatment of diabetic atherosclerosis from an oxidative stress/inflammatory perspective seems to be a more effective modality to prevent and delay plaque formation and progression. This study aimed to evaluate the effects of l-limonene (LMN) on oxidative stress/inflammatory responses in the aortic artery of diabetic atherosclerosis-modeled rats. Male Wistar rats (n = 30, 250-280 g, 12 weeks old) were used to establish a diabetic atherosclerosis model (8 weeks) using high-fat diet/low-dose streptozotocin. LMN (200 mg/kg/day) was administered orally, starting on day 30th before tissue sampling. Plasma lipid profiles, aortic histopathological changes, atherogenic index, aortic artery levels of oxidative stress markers (manganese superoxide dismutase, glutathione, and 8-isoprostane), inflammatory markers (tumor necrosis factor-alpha, interleukin (IL)-6, and IL-10), and expression of phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK)/AMPK, Sirtuin 1 (SIRT1), and p-p65/p65 proteins were evaluated. The administration of LMN to diabetic rats improved lipid profiles, aortic histopathological morphology, and atherogenic index (P < 0.05 to P < 0.001). It also increased enzymatic antioxidant activities, decreased 8-isoprostane level, suppressed inflammatory response, upregulated p-AMPK and SIRT1 proteins, and downregulated p-p65 protein (P < 0.05 to P < 0.01). Inhibiting the AMPK through the administration of compound C significantly abolished or reversed the positive effects of LMN in diabetic rats (P < 0.05 to P < 0.01). LMN treatment had dual anti-oxidative and anti-inflammatory actions against atherosclerosis in the aortic artery of diabetic rats. Atheroprotection by LMN was mediated partly through modulation of AMPK/SIRT1/p65 nuclear factor kappa B signaling pathway. LMN appears to be a promising anti-atherosclerotic modality to improve the quality of life in diabetic patients.

Limonene, a Monoterpene, Mitigates Rotenone-Induced Dopaminergic Neurodegeneration by Modulating Neuroinflammation, Hippo Signaling and Apoptosis in Rats.

Rotenone (ROT) is a naturally derived pesticide and a well-known environmental neurotoxin associated with induction of Parkinson's disease (PD). Limonene (LMN), a naturally occurring monoterpene, is found ubiquitously in citrus fruits and peels. There is enormous interest in finding novel therapeutic agents that can cure or halt the progressive degeneration in PD; therefore, the main aim of this study is to investigate the potential neuroprotective effects of LMN employing a rodent model of PD measuring parameters of oxidative stress, neuro-inflammation, and apoptosis to elucidate the underlying mechanisms. PD in experimental rats was induced by intraperitoneal injection of ROT (2.5 mg/kg) five days a week for a total of 28 days. The rats were treated with LMN (50 mg/kg, orally) along with intraperitoneal injection of ROT (2.5 mg/kg) for the same duration as in ROT-administered rats. ROT injections induced a significant loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and DA striatal fibers following activation of glial cells (astrocytes and microglia). ROT treatment enhanced oxidative stress, altered NF-κB/MAPK signaling and motor dysfunction, and enhanced the levels/expressions of inflammatory mediators and proinflammatory cytokines in the brain. There was a concomitant mitochondrial dysfunction followed by the activation of the Hippo signaling and intrinsic pathway of apoptosis as well as altered mTOR signaling in the brain of ROT-injected rats. Oral treatment with LMN corrected the majority of the biochemical, pathological, and molecular parameters altered following ROT injections. Our study findings demonstrate the efficacy of LMN in providing protection against ROT-induced neurodegeneration.

D-Limonene Promotes Anti-Obesity in 3T3-L1 Adipocytes and High-Calorie Diet-Induced Obese Rats by Activating the AMPK Signaling Pathway.

D-limonene (LIM) is a common monoterpene compound, principally found in citrus essential oils. This study investigated the anti-obesity effect of LIM on the 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway in 3T3-L1 adipocytes and high-calorie diet-induced obese rats and confirmed the optimally effective dose of LIM. The 3T3-L1 adipocytes were treated with 0.05−0.4 mg/mL LIM for 10 days and oil red O and triglyceride (TG) content were used to determine the levels of lipid accumulation. The results showed that more than 0.05 mg/mL LIM inhibited lipid accumulation by reducing oil red O in 3T3-L1 adipocytes. Masses of 0.2 and 0.4 mg/mL LIM also decreased the TG contents in 3T3-L1 adipocytes. On the other hand, Wistar rats were given high-calorie diets, combined with LLIM (154 mg/kg) and HLIM (1000 mg/kg) treatments, for 16 weeks. The result shows that LLIM and HLIM decreased body weight, total fat tissue weight, and serum low-density lipoprotein-cholesterol (LDLc) levels. HLIM reduced serum TG and increased serum lipase and high-density lipoprotein-cholesterol (HDLc) levels. Moreover, the anti-obesity metabolic pathway showed that LIM (>0.05 mg/mL) in 3T3-L1 adipocytes and LIM (>154 mg/kg) in high-calorie diet-induced obese rats could activate the AMPK signaling pathway. The activated AMPK regulated the mRNA expression related to adipogenesis (PPARγ, C/EBPα, FABP4), lipogenesis (SREBP-1c, ACC, FAS), and lipolysis (ATGL, HSL) to inhibit obesity. This finding demonstrates that LIM has anti-obesity properties. Namely, it is seen that LIM acts by regulating the AMPK signaling pathway in 3T3-L1 adipocytes and high-calorie diet-induced obese rats. In terms of dose−response, LIM (154 mg/kg) would be an optimal effective dose for anti-obesity induced by a high-calorie diet.

Effect of D-Limonene Nanoemulsion Edible Film on Banana (Musa sapientum Linn.) Post-Harvest Preservation.

D-limonene (4-isopropenyl-1-methylcyclohexene) is an important compound in several citrus essential oils (such as orange, lemon, tangerine, lime, and grapefruit). It has been used as a flavoring agent and as a food preservative agent, with generally recognized as safe (GRAS) status. D-limonene has been well-studied for its anti-inflammatory, antioxidant, anti-cancer, and antibacterial properties. The antibacterial activity of D-limonene against food-borne pathogens was investigated in this study by preparing a D-limonene nanoemulsion. The D-limonene solution and nanoemulsion have been prepared in six concentrations, 0.04%, 0.08%, 0.1%, 0.2%, 0.4%, and 0.8% (v/v), respectively, and the antibacterial activity was tested against four food-borne pathogens (Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica, and Escherichia coli). The results showed that the D-limonene nanoemulsion had good nanoscale and overall particle size uniformity, and its particle size was about 3~5 nm. It has been found that the D-limonene solution and nanoemulsion have a minimal inhibitory concentration of 0.336 mg/mL, and that they could inhibit the growth of microorganisms efficiently. The data indicate that the D-limonene nanoemulsion has more antibacterial ability against microorganisms than the D-limonene essential oil. After bananas are treated with 1.0% and 1.5% D-limonene nanoemulsion coatings, the water loss of the bananas during storage and the percentage of weight loss are reduced, which can inhibit the activity of pectinase. The application of a biocoating provides a good degree of antibacterial activity and air and moisture barrier properties, which help with extending the shelf life of bananas.

Antioxidative, anticancer, and antibacterial activities of a nanogel containing Mentha spicata L. essential oil and electrospun nanofibers of polycaprolactone-hydroxypropyl methylcellulose.

BACKGROUND: As the largest organ, the skin has been frequently affected by trauma, chemical materials, toxins, bacterial pathogens, and free radicals. Recently, many attempts have been made to develop natural nanogels that, besides hydrating the skin, could also be used as antioxidant or antibacterial agents. METHODS: In this study, the chemical composition of the Mentha spicata essential oil was first investigated using GC-MS analysis. Its nanoemulsion-based nanogel was then investigated; successful loading of the essential oil in the nanogel was confirmed using FTIR analysis. Besides, nanogel's antioxidative, anticancer, and antibacterial activities were investigated. RESULTS: Carvone (37.1%), limonene (28.5%), borneol (3.9%), ß-pinene (3.3%), and pulegone (3.3%) were identified as five major compounds in the essential oil. By adding carboxymethylcellulose (3.5% w/v) to the optimal nanoemulsion containing the essential oil (droplet size of 196 ± 8 nm), it was gelified. The viscosity was fully fitted with a common non-Newtonian viscosity regression, the Carreau-Yasuda model. The antioxidant effect of the nanogel was significantly more potent than the essential oil (P < 0.001) at all examined concentrations (62.5-1000 µg/mL). Furthermore, the potency of the nanogel with an IC50 value of 55.0 µg/mL was substantially more (P < 0.001) than the essential oil (997.4 µg/mL). Also, the growth of Staphylococcus aureus and Escherichia coli after treatment with 1000 µg/mL nanogel was about 50% decreased compared to the control group. Besides, the prepared electrospun polycaprolactone-hydroxypropyl methylcellulose nanofibers mat with no cytotoxic, antioxidant, or antibacterial effects was proposed as lesion dressing after treatment with the nanogel. High potency, natural ingredients, and straightforward preparation are advantages of the prepared nanogel. Therefore, it could be considered for further consideration in vivo studies.

Proximal discrepancy in intrinsic atomic interaction arrests G2/M phase by inhibiting Cyclin B1/CDK1 to infer molecular and cellular biocompatibility of D-limonene.

The quest for different natural compounds for different biomedical applications especially in the treatment of cancer is at a high pace with increasing incidence of severity. D-limonene has been portrayed as one of the effective potential candidate centered to the context of breast cancer. The anticipation of its count as an effective biomedical agent required a detailed understanding of their molecular mechanism of biocompatibility. This study elucidates the mechanistic action of D-limonene channelized by the induction of apoptosis for controlling proliferation in breast cancer cells. The possible mechanism was explored through an experimental and computational approach to estimate cell proliferation inhibition, cell cycle phase distribution, apoptosis analysis using a flow cytometry, western blotting and molecular docking. The results showed reduced dose and time-dependent viability of MCF7 cells. The study suggested the arrest of the cell cycle at G2/M phase leading to apoptosis and other discrepancies of molecular activity mediated via significant alteration in protein expression pattern of anti-apoptotic proteins like Cyclin B1 and CDK1. Computational analysis showed firm interaction of D-limonene with Cyclin B1 and CDK1 proteins influencing their structural and functional integrity indicating the mediation of mechanism. This study concluded that D-limonene suppresses the proliferation of breast cancer cells by inducing G2/M phase arrest via deregulation of Cyclin B1/CDK1.

Role of the major terpenes of Callistemon citrinus against the oxidative stress during a hypercaloric diet in rats.

1,8-Cineole, limonene and α-terpineol are the major terpenes present in Callistemon citrinus. This study reports for the first time that terpenes attenuate the oxidative stress in rats fed with high-fat-sucrose diet (HFSD) via antioxidant and anti-inflammatory mechanisms. Thirty-six male Wistar rats were divided into six groups (n = 6). Control (fed standard food, HFSD (fed with 41.7% fat and 16.6% sucrose), HFSD + 1,8-cineole (0.88 mg/kg body weight), limonene (0.43 mg/kg body weight), α-terpineol (0.32 mg/kg body weight) and a mixture of the three terpenes, given daily by gavage for 15 weeks. Morphometric and biochemical parameters were taken. Paraoxonase (PON1), reduced glutathione (GSH), lipid peroxidation products malondialdehyde (MDA) and hydroxyalkenals (HNE), advanced oxidation protein products (AOPP) and pro-inflammatory cytokines were measured in liver homogenates. All terpenes showed a remarkable reduction in weight gain, fat deposition, serum glucose and, triacylglycerol levels. However, terpenes presented different effects on the hepatic cell and the oxidative biomarkers. Conversely, the three terpenes and the mixture showed the same positive effect on the tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), leptin and adiponectin levels. Finally, 1,8-cineole, limonene and α-terpineol demonstrate significant anti-inflammatory effects and differential effects on the oxidative stress, suggesting the importance of these terpenes in Callistemon citrinus activities.

Cytotoxic Effect of the Essential oils from Erigeron Canadensis L. on Human Cervical Cancer HeLa Cells in Vitro.

Erigeron Canadensis L. (E. canadensis) is a widely distributed invasive weed species in China. Potentially anti-cancer qualities may exist in its essential oils (EOs). The purpose of this study was to analyze the components of the EOs of E. canadensis and their effects on the normal liver cell lines L02 and the human cervical cancer cell lines HeLa. The EOs from the upper region of E. canadensis were prepared, its components were identified by GC/MS. Cell viability, cell morphology observation, AO/EB dual fluorescence staining assay, flow cytometry, mitochondrial membrane potential, western blot, caspase inhibitor test, and oxidative stress tests were used to investigate the impact of the EOs on HeLa cells. Network pharmacological analysis was employed to study the potential mechanism of the EOs in the treatment of cervical cancer. According to the findings, the EOs had 21 chemical components, of which limonene made up 65.68 %. After being exposed to the EOs, the cell viability of HeLa and L02 dramatically declined. The inhibition of EOs was more effective than that of limonene when used in an amount equivalent to that in the EOs. L02 cells were less susceptible to the cytotoxicity of EOs than HeLa cells were. Furthermore, EOs altered the cell cycle in HeLa cells and caused oxidative stress and apoptosis. Compared with the control group, the reactive oxygen species (ROS) levels increased in HeLa cells at first and then decreased, total superoxide dismutase (SOD) and catalase (CAT) activities in HeLa cells significantly decreased. G1 phase cells decreased whereas G2/M phase cells increased. The rate of apoptosis rose. Reduced mitochondrial membrane potential and Caspase-3, -9, and -12 protein expression were both observed. Nerolidol, dextroparaffinone, and α-pinene were shown to be the primary components for the suppression of HeLa cells, according to the results of the prediction of pharmacologic targets. In conclusion, findings of this study indicated the EOs may have the potential to curb the growth of cervical cancer cells. Further research is needed to explore the in vivo effect of EOs.