Menthol Treatment Research Articles

Menthol-Based Extraction of Fragile Wooden Coffin Lid (7–10th Centuries CE) in Laboratory Archaeology Excavation

Block lifting is a key step in stabilizing and removing fragile remains at archaeological excavation sites. Due to its favorable working properties and adhesive effect, menthol has recently been proposed as a volatile binding medium for temporary consolidation in archaeological conservation. This paper presents a case study on the use of menthol in the extraction and restoration of a large wooden coffin lid, approximately 1.9 m long and 0.9 m wide, from tomb 11 (M11) at Xie’ertala, located east of a Xie’ertala town in Hailar City, Inner Mongolia, dating to the 7th to 10th centuries CE. This coffin lid had fragmented into numerous wooden pieces, and was preserved in a relatively arid steppe environment, necessitating the extraction of the lid as a consolidated block. The use of menthol for consolidating and lifting the highly fragmented wooden coffin lid was intended to preserve critical archaeological information while avoiding damage to the underlying objects. An analysis of the physicochemical properties of these wooden remains suggests that the timber used for the coffin lid belongs to a common pine species from the Hulunbuir region. The degradation of the coffin lid was relatively mild, as shown by Fourier Transform Infrared Spectroscopy (FT-IR) and Scanning Electron Microscope (SEM) results. Dynamic Vapor Sorption (DVS) tests indicated that the hygroscopicity of the archaeological wood was 23.4%, compared to 21.1% for the reference sample, demonstrating good environmental stability. The safety of menthol as a treatment for fragile wooden remains was evaluated by comparing changes in the morphological and porosity characteristics of the coffin lid before and after menthol treatment. After treatment, the widths of the fissures remained largely unchanged, with all relative variations being less than 1%, and the porosity as well as pore size distribution of the wood showed negligible changes. Gas Chromatography–Mass Spectrometry (GC-MS) results showed that only 0.6% of menthol residue remained after 8 days of sublimation. This pilot study demonstrates that menthol is a safe temporary consolidant for block lifting and offers a promising alternative to the widely used cyclododecane. In conclusion, this research provided a new approach for conservators to safely lift similarly large and fragile wood remains during archaeological excavations.

Therapeutic potential of monoterpene molecules acts against 7KCh-mediated oxidative stress and neuroinflammatory amyloidogenic signalling pathways

Alzheimer’s disease (AD) is a degenerative disorder characterised by amyloid-beta aggregates activated by the accumulation of lipid molecules and their derivatives, especially 7-ketocholesterol (7KCh), an oxidised lipid that plays a great part in the progression of AD. The current therapeutics need bio-potential molecules and their biomedical application preventing 7KCh-induced cytotoxicity. In this study, bornyl acetate (BA) and menthol (ME), the natural monoterpenes were investigated for their neuroprotective effects against 7KCh-induced SH-SY5Y cells and their effects were compared to the standard drug galantamine (GA). 7KCh-induced changes like lipid accumulation, amyloid generation, free radical generation, acetylcholinesterase levels, calcium accumulation and mitochondrial membrane integrity were analysed in SH-SY5Y cells with or without BA and ME treatment. Furthermore, various mediators involved in the amyloidogenic, inflammatory and apoptotic pathways were studied. In our results, the cells induced with 7KCh upon co-treatment with BA and ME significantly reduced lipid accumulation and amyloid generation through toll-like receptor (TLR) 4 suppression and enhanced ATP binding cassette (ABCA) 1-mediated clearance. Co-treatment with BA and ME concurrently regulated oxidative stress, acetylcholinesterase activity, mitochondrial membrane potential and intracellular calcification altered by 7KCh-induced SH-SY5Y cells. Moreover, 7KCh-induced cells showed elevated mRNA levels of misfolded protein markers and apoptotic mediators which were significantly downregulated by BA and ME co-treatment. In addition, the protein expression of amyloidogenic, proinflammatory as well as pro-apoptotic markers was decreased by BA and ME co-treatment in 7KCh-induced cells. Overall, BA and ME mediated inhibition of amyloidogenic activation and cell survival against 7KCh-induced inflammation, thereby preventing the onset and progression of AD in comparison to GA.

Protective effect of menthol against thioacetamide-induced hepatic encephalopathy by suppressing oxidative stress and inflammation, augmenting expression of BDNF and α7-nACh receptor, and improving spatial memory

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome that can occur in people with acute or chronic liver disease. Here, we investigated the effects of menthol, a natural monoterpene, on HE induced by thioacetamide (TA) in male Wistar rats. The rats received 200 mg/kg of TA twice a week for four weeks and were administered 10 mg/kg of menthol intraperitoneally daily for the same period. The results showed that menthol treatment reduced oxidative stress and inflammation in the livers and hippocampi of the rats that received TA. It also lowered the levels of ammonium and liver enzymes AST, ALT, ALP, and GGT in the serum of these animals and prevented liver histopathological damage. In addition, the expression and activity of acetylcholinesterase in the hippocampus of HE model rats were decreased by menthol. Likewise, this monoterpene reduced the expression of TLR4, MyD88, and NF-κB in the hippocampus while increasing the expression of BDNF and α7-nACh receptor. Menthol also reduced neuronal death in the hippocampal cornu ammonis-1 and dentate gyrus regions and reduced astrocyte swelling, which led to improved learning and spatial memory in rats with HE. In conclusion, the study suggests that menthol may have strong protective effects on the liver and brain, making it a potential treatment for HE and neurodegenerative diseases.

Topical menthol for chemotherapy-induced peripheral neuropathy: a randomised controlled trial in breast cancer

ObjectivesChemotherapy-induced peripheral neuropathy (CIPN) symptom is one of the side effects of paclitaxel in breast cancer patients. This randomised controlled study was conducted to investigate the effect of topical menthol...

#788 Mitigation of tubular apoptosis and cystogenesis in ADPKD: the impact of TRPM8 through restriction of mitochondrial function

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD), characterized by numerous fluid-filled cysts and a progressive decline in renal function, is a prevalent hereditary kidney disorder. Approximately 70% of individuals affected by ADPKD may develop end-stage renal disease, typically manifesting around the median age of 58 years. Despite its prevalence, effective clinical treatments for ADPKD remain elusive. Transient receptor potential melastatin member 8 (TRPM8), serving as a sensor for cold temperatures and activated by the cooling agent menthol, orchestrates cellular Ca2+ signaling by facilitating Ca2+ entry into the cytosol. However, the role of TRPM8 in ADPKD remains undefined. Method The WT 9-12, an epithelial cell line isolated from the proximal tubule of a patient with ADPKD, was utilized for in vitro experiments. Eight-week-old wild-type (WT), Pkd1 knockdown (pkd1kd), and Trpm8, Pkd1 double mutant (T-/-P) mice were used for experiments. Renal Trpm8 was activated by administration of a 0.5% menthol diet for 7 months in mice. The cyst index was measured by H&E stain and calculated using Image J. Apoptosis was evaluated by TUNEL assay (In Situ Cell Death Detection Kit, TMR red). Protein and mRNA expression of mice kidneys was detected by Western blotting and qRT-PCR, respectively. Blood urea nitrogen (BUN) was measured by HITACHI 7600 analyzer. Results The activation of TRPM8 by menthol treatment resulted in an elevation of intracellular Ca2+ levels, subsequently impeding proliferation in WT9-12 cells. Consistently, Trpm8 activation suppressed kidney weight, kidney weight-to-body weight ratio, BUN, fibrosis, and cystogenesis in Pkd1kd mice, while no such effects were observed in WT and T-/-P mice. This suggests a protective role of Trpm8 in ADPKD. Mechanistically, Trpm8 activation mitigated tubular apoptosis in the kidneys of Pkd1kd mice, as evidenced by reduced TUNEL-positive cells and downregulated Bax and cleaved caspase-3, with no such effects observed in WT and T-/-P mice. Intriguingly, the proliferation-associated signaling pathway, ERK, was also suppressed by Trpm8. Proteomic analysis of mice kidneys revealed that Trpm8 regulates mitochondrial dysfunction, EIF2 and Sirtuin signaling pathways. The result was verified in WT9-12 cells showing reduced mitochondrial respiration, particularly impacting oxidative phosphorylation by TRPM8. Conclusion Trpm8 inhibits tubular apoptosis and cyst formation through the restriction of mitochondrial function, subsequently retarding ADPKD development in mice. These findings illuminate a novel and promising strategy for intervening in ADPKD.

Effect of Menthol Treatment on the Sprouting and Quality of Potato Tuber

This study used Dutch potatoes at the end of dormancy as a material to explore the impact of menthol (0.2 and 0.5 g/kg based on potato mass) treatment on sprouting inhibition and potato quality. The findings revealed that a menthol concentration of 0.5 g/kg effectively inhibited potato tuber sprouting and significantly reduced glucoside alkaloid production. After a storage period of 15 days, the sprouting percentage and glucoside alkaloid content of potatoes treated with 0.5 g/kg menthol were observed to be significantly lower at 4.17% and 68.63 mg/kg, respectively, compared to the control group which exhibited values of 100% and 282.01 mg/kg, respectively (p < 0.05). Throughout the storage period, 0.5 g/kg of menthol promoted respiration, reduced malondialdehyde production in potatoes, inhibited polyphenol oxidase activity, and slowed down tissue browning. Additionally, it mitigated the decline in starch and soluble protein content, inhibiting the accumulation of reducing sugars.

Therapeutic Effects of Oral Application of Menthol and Extracts from Tormentil (Potentilla erecta), Raspberry Leaves (Rubus idaeus), and Loosestrife (Lythrum salicaria) during Acute Murine Campylobacteriosis.

Human food-borne infections with the enteropathogen Campylobacter jejuni are becoming increasingly prevalent worldwide. Since antibiotics are usually not indicated in campylobacteriosis, alternative treatment regimens are important. We here investigated potential disease-alleviating effects of menthol and of extracts from tormentil, raspberry leaves, and loosestrife in acute murine campylobacteriosis. Therefore, C. jejuni-infected microbiota-depleted IL-10-/- mice were orally treated with the compounds alone or all in combination from day 2 until day 6 post-infection. Whereas neither treatment regimen affected gastrointestinal pathogen loads, the combination of compounds alleviated C. jejuni-induced diarrheal symptoms in diseased mice on day 6 post-infection. Furthermore, the therapeutic application of tormentil and menthol alone and the combination of the four compounds resulted in lower colonic T cell numbers in infected mice when compared to placebo counterparts. Notably, pro-inflammatory cytokines measured in mesenteric lymph nodes taken from C. jejuni-infected mice following tormentil, menthol, and combination treatment did not differ from basal concentrations. However, neither treatment regimen could dampen extra-intestinal immune responses, including systemic pro-inflammatory cytokine secretion on day 6 post-infection. In conclusion, the combination of menthol and of extracts from tormentil, raspberry leaves, and loosestrife constitutes an antibiotic-independent approach to alleviate campylobacteriosis symptoms.

Abstract P229: Beyond Cooling Sensation: Topical Penile Administration Of The Trpm8 Agonist Improves Hypertension Associated-erectile Dysfunction

Background and Purpose: Erectile dysfunction (ED) is an indicator for futurecardiovascular events. The search for new targets is crucial due to the low efficacy ofPDE5 inhibitors in patients with endothelial dysfunction. Menthol, a TRPM8 agonist, iswidely used in preparations to improve sensation during sexual intercourse, although nodata are reported for the improvement of ED. So, we hypothesize that chronic topicalactivation of TRPM8 improves hypertension associated-ED. Experimental Approach: Adult (9-12 weeks) male spontaneously hypertensive rats (SHR) and Wistar rats wereused for protein expression analysis and TRPM8 agonist curves in isolated CC. We alsotreated rats topically on the penis for 30 days with carbopol 940 (hydrogel, vehicle) ormenthol (carbopol 940 + menthol 0.1%). After treatment, we measured the mean bloodpressure (MBP), CC and iliac (IL) artery contractility. Key Results: Both strains of rathad similar TRPM8 expression in CC by western blotting and immunofluorescence.TRPM8 activation by cooling compounds, icilin (10 -8 –10 -4 M) or menthol (10 -7 –10 -3 M),relaxed CC strips pre-contracted by phenylephrine (PE, 10 -6 or 10 -5 M), but with reducedeffect in SHR. Interestingly, the topical penile treatment with menthol decreased the MBPof the SHR (151.8±6.8 mmHg, n=6) compared to the SHR vehicle group (174.6±2.6mmHg, n=5; p<0.05). In addition, in CC, the concentration-response curves to PE andthromboxane agonists were shifted to the left after menthol treatment. Interestingly, nochange in acetylcholine and sodium nitroprusside (SNP) relaxation were observed aftertopical penile treatment with menthol. The concentration-response curves to PE and SNPwere shifted to the left in IL arterial rings. This suggests topical menthol administrationinduced systemic vascular influence leading to a response like the normotensive Wistar. Conclusion: TRPM8 channels are expressed and functional in the CC, with decreasedsensitivity in SHR. Also, our data suggest that topical administration of menthol improvesthe reactivity of vascular tissue, demonstrating the potential of TRPM8 activation fortherapeutic benefits in hypertension associated-ED.

Influence of topical menthol gel on thermoregulation and perception while walking in the heat.

Menthol is known to elicit opposing thermoregulatory and perceptual alterations during intense exercise. The current purpose was to determine the thermoregulatory and perceptual effects of topical menthol application prior to walking in the heat. Twelve participants walked (1.6m s-1, 5% grade) for 30min in the heat (38°C, 60% relative humidity) with either a 4% menthol or control gel on the upper (shoulder to wrist) and lower (mid-thigh to ankle) limbs. Skin blood flow (SkBF), sweat (rate, composition), skin conductivity, heart rate, temperature (skin, core), and thermal perception were measured prior to and during exercise. Skin conductivity expressed as time to 10, 20, 30, and 40 µS was delayed due to menthol (559 ± 251, 770 ± 292, 1109 ± 301, 1299 ± 335s, respectively) compared to the control (515 ± 260, 735 ± 256, 935 ± 300, 1148 ± 298s, respectively, p = 0.048). Sweat rate relative to body surface area was lower due to menthol (0.55 ± 0.16 L h-1 m(2)-1) than the control (0.64 ± 0.16 L h-1 m(2)-1, p = 0.049). Core temperature did not differ at baseline between the menthol (37.4 ± 0.3°C) and control (37.3 ± 0.4°C, p = 0.298) but was higher at 10, 20, and 30 min due to menthol (37.5 ± 0.3, 37.7 ± 0.2, 38.1 ± 0.3°C, respectively) compared to the control (37.3 ± 0.4, 37.4 ± 0.3, 37.7 ± 0.3°C, respectively, p < 0.05). The largest rise in core temperature from baseline was at 30min during menthol (0.7 ± 0.3°C) compared to the control (0.4 ± 0.2°C, p = 0.004). Overall, the menthol treatment was perceived cooler, reaching "slightly warm" whereas the control treatment reached "warm" (p < 0.001). Menthol application to the limbs impairs whole-body thermoregulation while walking in the heat despite perceiving the environment as cooler.

Irritant-evoked reflex tachyarrhythmia in spontaneously hypertensive rats is reduced by inhalation of TRPM8 agonists l-menthol and WS-12.

Inhalation of noxious irritants activates nociceptive sensory afferent nerves innervating the airways, inducing reflex regulation of autonomic networks and the modulation of respiratory drive and cardiovascular (CV) parameters such as heart rate and blood pressure. In healthy mammals, irritant-evoked pulmonary-cardiac reflexes cause parasympathetic-mediated bradycardia. However, in spontaneously hypertensive (SH) rats, irritant inhalation also increases sympathetic drive to the heart. This remodeled pulmonary-cardiac reflex may contribute to cardiovascular risk caused by inhalation of air pollutants/irritants in susceptible individuals with cardiovascular disease (CVD). Previous studies have shown that the cooling mimic l-menthol, an agonist for the cold-sensitive transient receptor potential melastatin 8 (TRPM8), can alleviate nasal inflammatory symptoms and respiratory reflexes evoked by irritants. Here, we investigated the impact of inhalation of TRPM8 agonists l-menthol and WS-12 on pulmonary-cardiac reflexes evoked by inhalation of the irritant allyl isothiocyanate (AITC) using radiotelemetry. l-Menthol, but not its inactive analog d-menthol, significantly reduced the AITC-evoked reflex tachycardia and premature ventricular contractions (PVCs) in SH rats but had no effect on the AITC-evoked bradycardia in either SH or normotensive Wistar-Kyoto (WKY) rats. WS-12 reduced AITC-evoked tachycardia and PVCs in SH rats, but this more potent TRPM8 agonist also reduced AITC-evoked bradycardia. l-Menthol had no effect on heart rate when given alone, whereas WS-12 evoked a minor bradycardia in WKY rats. We conclude that stimulation of TRPM8-expressing afferents within the airways reduces irritant-evoked pulmonary-cardiac reflexes, especially the aberrant reflex tachyarrhythmia in SH rats. Airway menthol treatment may be an effective therapy for reducing pollution-associated CV exacerbations.NEW & NOTEWORTHY Irritant-evoked pulmonary-cardiac reflexes are remodeled in spontaneously hypertensive (SH) rats-causing de novo sympathetic reflexes that drive tachyarrhythmia. This remodeling may contribute to air pollution-associated risk in susceptible individuals with cardiovascular disease. We found that inhalation of TRPM8 agonists, l-menthol and WS-12, but not the inactive analog d-menthol, selectively reduces the reflex tachyarrhythmia evoked by allyl isothiocyanate (AITC) inhalation in SH rats. Use of menthol may protect susceptible individuals from pollution-associated CV exacerbations.

Underlying mechanism of menthol on controlling postharvest citrus sour rot caused by Geotrichum citri-aurantii

Sour rot, caused by Geotrichum citri-aurantii, has affected fruit quality and caused severe economic losses. Menthol is widely applied to medicine and food industries due to its antiseptic and antimicrobial effects, whereas its application in postharvest fruit management is rarely reported. The present study investigated the antifungal activity of menthol and its underlying mechanism involved. The in vitro experiments showed that the minimum inhibitory concentration (MIC) of menthol was 6.4 mmol L-1. Menthol induced the formation and accumulation of reactive oxygen species (ROS) in G. citri-aurantii and triggered severe membrane lipid peroxidation, resulting in the destruction of plasma membrane integrity. The activity of extracellular alkaline phosphatase (AKP) was increased after menthol treatment. Otherwise, the decrease of blue fluorescence in the cell wall and the change of cell wall morphology were also observed, indicating that the cell wall structure was disrupted. Notably, in vivo, the disease incidence of citrus fruit decreased by 43% after the application of 128 mmol L-1 menthol. Furthermore, menthol treatment maintained the quality of citrus fruit and enhanced defense-related enzyme activities in citrus. To further explore the potential pathogenicity of G. citri-aurantii, we de novo assembled the genome of G. citri-aurantii G50 based on Illumina NovaSeq PE150 and PacBio Sequel. The revised genome size was 25.41 Mb, assembled onto 27 scaffolds with a total length of 27,548,716 bp, 38.49% GC content and 4181 protein-coding genes. Herein, we identified four CWI-MAPK-related genes (GciPKC1, GciBCK1, GciMKK1 and GciSLT2). The expressions of these genes were down-regulated after menthol treatment, influencing on downstream chitin and glucan synthesis. Collectively, these results demonstrated that menthol might be beneficial as a potential alternative for decay management of citrus sour rot. Our research provided new perspectives for postharvest disease control and management control of this pathogen and elucidated its pathogenic mechanism.

Protective effects of menthol against sepsis-induced hepatic injury: Role of mediators of hepatic inflammation, apoptosis, and regeneration.

Liver dysfunction in sepsis is a major complication that amplifies multiple organ failure and increases the risk of death. Inflammation and oxidative stress are the main mediators in the pathophysiology of sepsis. Therefore, we investigated the role of menthol, a natural antioxidant, against sepsis-induced liver injury in female Wistar rats. Sepsis was induced by cecal ligation and puncture (CLP). Menthol (100 mg/kg) was given intragastric 2 h after CLP. Blood samples and liver tissues were collected 24 h after surgery. Menthol significantly (p < 0.05) attenuated the sepsis-induced elevation in serum liver enzymes and improved the hepatic histopathological changes. Menthol treatment significantly (p < 0.05) decreased hepatic levels of tumor necrosis factor-alpha, malondialdehyde, total nitrite, and cleaved caspase-3. It restored the hepatic levels of superoxide dismutase and reduced glutathione. Additionally, menthol significantly (p < 0.05) increased hepatic levels of B-cell lymphoma 2 (Bcl-2); an anti-apoptotic factor, and proliferating cell nuclear antigen (PCNA), a biomarker of regeneration and survival. Our results showed the therapeutic potential of menthol against liver injury induced by sepsis.

Activation of Cold‐Sensitive Afferents Inhibits Aberrant Irritant‐evoked Cardiopulmonary Reflexes in the Spontaneously Hypertensive (SH) Rat

Inhalation of irritants activates airway vagal afferent nerves, evoking cardiopulmonary reflexes. Evidence suggests that cardiopulmonary reflexes evoked by irritant/pollution inhalation are remodeled from bradycardia in normal subjects to tachycardia and ventricular arrhythmia in patients with pre‐existing cardiovascular disease. We have shown similar remodeling in the spontaneously hypertensive (SH) rat: selective activation of airway vagal afferent nerves via inhalation of the TRPA1‐specific agonist allyl isothiocyanate (AITC) evoked parasympathetic‐mediated reflex bradycardia in normotensive wistar‐kyoto rats but evoked sympathetically‐mediated tachycardia and premature ventricular contractions (PVCs) in spontaneously hypertensive (SH) rats.Activation of TRPA1‐expressing airway vagal afferents also evokes cough in some conscious animal models. Cough reflexes have been shown to be inhibited by nasal stimulation with L‐menthol via activation of TRPM8 ion channels expressed on cold‐sensitive trigeminal afferents which regulate brainstem respiratory networks. Here, we investigated the cardiopulmonary reflexes evoked by inhalation of L‐menthol (150mg/ml) alone and in combination with AITC (4.7mg/ml) in SH rats. L‐menthol inhalation alone had no effect on respiration/heart rate, but, when inhaled during AITC inhalation, L‐menthol selectively inhibited the AITC‐evoked tachyarrhythmia and tachypnea. Importantly, the inactive isomer D‐menthol (150mg/ml) failed to affect AITC‐evoked sympathoexcitatory reflexes. In another set of studies, the selective TRPM8 agonist WS‐12 (300mM) also inhibited the AITC‐evoked tachyarrhythmia. Therefore, we conclude that aberrant cardiopulmonary reflexes evoked by irritant inhalation are reduced by activation of menthol‐sensitive pathways within the airways. This data supports the therapeutic potential of menthol treatment against irritant‐evoked cardiopulmonary reflexes.

Sauna-like conditions or menthol treatment reduce tau phosphorylation through mild hyperthermia

In Alzheimer's disease (AD), hyper-phosphorylation and aggregation of tau correlate with clinical progression and represent a valid therapeutic target. A recent 20-year prospective study revealed an association between moderate to high frequency of Finnish sauna bathing and a lower incidence of dementia and AD, but the molecular mechanisms underlying these benefits remain uncertain. Here, we tested the hypothesis that sauna-like conditions could lower tau phosphorylation by increasing body temperature. We observed a decrease in tau phosphorylation in wild-type and hTau mice as well as in neuron-like cells when exposed to higher temperatures. These effects were correlated with specific changes in phosphatase and kinase activities, but not with inflammatory or heat shock responses. We also used a drug strategy to promote thermogenesis: topical application of menthol, which led to a sustained increase in body temperature in hTau mice, concomitant with a significant decrease in tau phosphorylation. Our results suggest that sauna-like conditions or menthol treatment could lower tau pathology through mild hyperthermia, and may provide promising therapeutic strategies for AD and other tauopathies.

Menthol Targeting AMPK Alleviates the Inflammatory Response of Bovine Mammary Epithelial Cells and Restores the Synthesis of Milk Fat and Milk Protein.

Mastitis is one of the most serious diseases that causes losses in the dairy industry, seriously impairing milk production and milk quality, and even affecting human health. Menthol is a cyclic monoterpene compound obtained from the stem and leaves of peppermint, which has a variety of biological activities, including anti-inflammatory and antioxidant activity. The purpose of this study was to investigate the preventive effect of menthol on the lipopolysaccharide-induced inflammatory response in primary bovine mammary gland epithelial cells (BMECs) and its anti-inflammatory mechanism. First, BMECs were isolated and amplified from the udders of Holstein cows by enzymatic hydrolysis. BMECs were treated with menthol (10, 50, 100, 200 μM) for 1h, followed by lipopolysaccharide (5μg/ml) for 12 h. Lipopolysaccharide treatment upregulated the protein levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (INOS) and the mRNA abundance of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), while menthol was able to inhibit this effect. The inhibitory effect of menthol on proinflammatory factors was significantly reduced when autophagy was blocked using 3-Methyladenine (5μg/ml), an inhibitor of autophagy. Furthermore, lipopolysaccharide treatment reduced the expression levels of milk lipids and milk proteins, which were inhibited by menthol. In addition, menthol (200 μM) treatment was able to significantly upregulate the expression level of autophagy-related protein LC3B, downregulate the expression level of P62, promote the expression abundance of autophagy-related gene mRNA, and enhance significantly enhance autophagic flux. Interestingly, treatment of BMECs with menthol (200 μM) promoted the phosphorylation of AMP-activated protein kinase (AMPK) and unc-51 like kinase 1 (ULK1) and increased the nuclear localization of nuclear factor-E2 associated factor 2 (Nrf-2). When the AMPK pathway was blocked using compound C (10μg/ml), an inhibitor of AMPK, autophagy was significantly inhibited. Autophagy levels were significantly decreased after blocking the Nrf-2 pathway using ML385 (5μg/ml), an inhibitor of Nrf-2. Overall, the data suggest that menthol activates the AMPK-ULK1 pathway to initiate the onset of autophagy and maintains the level of autophagy through the AMPK-Nrf-2 pathway. In conclusion, the findings suggest that menthol may alleviate the inflammatory response in BMECs via the AMPK/ULK1/Nrf-2/autophagy pathway.

Menthol from Mentha piperita Suppresses the Milk Production of Lactating Mammary Epithelial Cells In Vivo and In Vitro.

Peppermint is traditionally used as an antigalactagogue in breastfeeding women. However, the suppressive mechanism remains unclear. The authors investigate whether and how peppermint influences milk production at the cellular and molecular levels. A lactating mammary epithelial cell (MEC) culture model that produces major milk components is prepared. Peppermint oil (PMO) suppresses β-casein production in conjunction with the induced enlargement of cytoplasmic lipid droplets (CLDs). PMO also significantly inactivates STAT5 and mTOR in the lactogenic signaling pathway. Menthol treatment, which is a primary PMO component, leads to decreases in β-casein production, enlarged CLDs, the inactivated STAT5 and mTOR. Eucalyptol and menthyl acetate, other components of peppermint, does not show suppressive effects on lactating MECs. The inactivation of STAT5 and mTOR upon menthol administration is also evident in alveolar MECs of lactating mice. Furthermore, lactating MECs expresses TRPM8 and TRPA1, which are menthol receptors known as cold receptors. Agonists of TRPM8 and TRPA1 suppresses β-casein production and inactivation of STAT5 and mTOR in the lactating MECs. These findings indicate that peppermint has potential as an antigalactagogue. Menthol is suggested to be an active compound in peppermint. TRPM8 and TRPA1 may function as receptors for menthol.

Reduced cytotoxicity in doxorubicin-exposed HepG2 cells pretreated with menthol due to upregulation of P-glycoprotein.

The aim of the present study was to examine changes in the expression and activity of P-glycoprotein (P-gp) in human hepatocellular carcinoma HepG2 cells after exposure to menthol, and their relationship to the cytotoxicity of and apoptotic responses to doxorubicin (DOX), a substrate of P-gp, in the cells. The expression of P-gp in HepG2 cells was significantly increased by menthol treatment. Intracellular accumulation of DOX in HepG2 cells was significantly lower in the menthol-treated group than in the control group, but this phenomenon was abolished in the presence of verapamil. Decreased cell viability by DOX was significantly attenuated by 24-h menthol treatment prior to DOX exposure, which coincided with the changes in mRNA expression of Bcl-xl and caspase-3. These results demonstrate that menthol causes hepatocellular carcinoma cells to acquire resistance to DOX by increasing its efflux through the upregulation of P-gp.

L‐Menthol Inhibits Cardiac Sympathoexcitation Caused by Inhaled Irritants in the Spontaneously Hypertensive Rat

Inhalation of irritants (e.g. particulate matter and ozone) triggers various efferent responses through activation of transient receptor potential ankyrin 1 (TRPA1) and TRP vanilloid 1 (TRPV1) channels expressed on airway vagal afferent nerves. In the airways they cause cough and mucus secretion and in conscious, healthy animals parasympathetic‐mediated reflexes include bradycardia and atrioventricular block. However, inhalation of air pollutants including particulate matter and ozone evokes sympathoexcitation (tachycardia, hypertension) in patients with cardiovascular disease. Interestingly, selective activation of airway vagal afferent nerves in spontaneously hypertensive (SH) rats via inhalation of the TRPA1‐specific agonist allyl isothiocyanate (AITC) evoked sympathetic‐mediated tachycardia and premature ventricular contractions (PVCs) consistent with reflexes evoked by inhaled pollution in patients with cardiovascular disease.Airway reflexes such as cough have been shown to be inhibited by nasal stimulation with L‐menthol via activation of cold‐sensitive trigeminal pathways which regulate brainstem respiratory networks. Here, we investigated the cardiac reflexes evoked by inhalation of L‐menthol (150mg/ml) alone and in combination with AITC (4.7mg/ml) in conscious SH rats. L‐menthol inhalation alone had no effect on heart rate, but, when inhaled during AITC inhalation, L‐menthol selectively inhibited the AITC‐evoked tachyarrhythmia. Inhibition of the AITC‐evoked sympathoexcitatory reflexes was not seen in identical studies performed with inactive D‐menthol (150mg/ml). Therefore, we conclude that pulmonary‐cardiac reflex tachyarrhythmia are reduced by activation of nasal menthol‐sensitive pathways. This data supports the potential for prevention of aberrant irritant‐evoked pulmonary‐cardiac reflexes by nasal menthol treatment.Support or Funding InformationNIH r56

Menthol relieves acid reflux inflammation by regulating TRPV1 in esophageal epithelial cells

Transient receptor potential cation channel subfamily V member 1 (TRPV1) plays an important role in pain and inflammatory responses. Previous studies have shown that the expression of TRPV1 increases in the sensory neurons of the esophagus during the development of gastroesophageal reflux disease and esophagitis, but the response of TRPV1 in esophageal epithelial cells (EECs), which directly confront the refluxed acid, is still unknown. Here, we found that acid reflux triggered esophageal damage, which was accompanied by increased expression of TRPV1 in EECs and TRPV1 channel activity in these cells. Furthermore, menthol inhibited the Ca2+ influx induced by acid stimulation in EECs. After menthol treatment, the expression of TRPV1 in EECs was significantly reduced, and their hyperplasia was significantly reduced; finally, the inflammation pathway elicited in EECs was diminished in mice with acid reflux. These results suggest that menthol improves the clinical symptoms caused by gastroesophageal acid reflux by interfering with TRPV1 in EECs.

Dietary Menthol Attenuates Inflammation and Cardiac Remodeling After Myocardial Infarction via the Transient Receptor Potential Melastatin 8.

Transient receptor potential melastatin subtype 8 (TRPM8) is a cold-sensing cation channel, mainly localized in the sensory neurons, which can be activated by menthol, a compound with a naturally cold sensation in mint. However, the effect of TRPM8 activation in inflammation and cardiac remodeling after myocardial infarction (MI) is not well defined. TRPM8 knockout (KO) mice (TRPM8-/-) and their wild-type littermates, aged 8 weeks, were randomly divided into sham and MI groups and were fed with chow or chow plus menthol. Dietary menthol significantly attenuated MI injury, evidenced by decreased survival rates and plasma cardiac troponion I levels, reduced infarct size and cardiomyocytes, declined collagen deposition, and rescued cardiac function and hemodynamics. However, these effects of menthol disappeared when mice were lacking TRPM8. Furthermore, feeding of menthol ameliorated elevated expression of inflammatory cytokines and chemokines, and aggravated inflammation infiltration in the MI mice but not in TRPM8-/- mice. In addition, menthol treatment increased the release of calcitonin gene-related peptide (CGRP), which were absent in TRPM8-/- mice. In conclusion, our results suggest that dietary menthol can protect against inflammation and cardiac remodeling after MI through activation of TRPM8.