Dysmenorrhea refers to severe pain in the lower abdomen and pelvis during menstruation [1
]. This condition affects over 90% of women worldwide [2
] and usually lasts for 24–72 h, and pain is the most critical symptom, which causes depression and a decline of quality of life [3
]. The cause of dysmenorrhea may be an increase in intracellular calcium influx in uterine smooth muscle cells. Calcium influx induces extracellular signal-regulated kinase (ERK1/2) and activates phosphor–myosin light chain 20 (p-MLC20) [4
]; furthermore, prostaglandin F2α (PGF2α) induces increased cyclooxygenase-2 (COX-2) [6
]. The increase of COX-2 may be due to the increased oxidative stress, which causes pain [7
]. The traditional treatment of primary dysmenorrhea is often in the form of non-steroidal anti-inflammatory drugs (NSAIDs), which are used to relieve pain or other accompanying symptoms [8
]. NSAIDs, including ibuprofen, aspirin, and naproxen, are used to exert anti-inflammatory and analgesic antipyretic effects by inhibiting COX-2, but they have several side effects, including increased headaches, dizziness, nausea, and indigestion. Over long-term usage, patients may develop drug resistance [11
As some serious side effects are present, researchers are eager to find a compound with a complementary effect to reduce the pain of this condition. Polyphenols, such as resveratrol and flavonoids in adlay hull extracts, can significantly reduce uterine contraction [12
]. It has been shown that polyphenols or flavonoids may play an important role in reducing uterine contraction and reducing the pain of dysmenorrhea.
Polyphenol compounds may be found in different plants and fruits and have a potential anti-oxidant ability and anti-inflammatory effect [14
]. The inhibition of oxidative stress is also related to the inhibition of uterine contraction [15
]. Recently, oleocanthal—a natural compound from extra virgin olive oil—has been found to be the polyphenolic compound that causes throat irritation in extra virgin olive oil, and it can achieve anti-inflammatory effects by inhibiting COX-2. Moreover, it has ibuprofen-like activity [17
], with a potential effect on pain-relief.
Extra virgin olive oil is one of the characteristic parts of the Mediterranean diet. It is produced in Spain, Italy, and Greece [18
]. Extra virgin olive oil is rich in polyphenolic compounds, including oleocanthal, oleuropein, oleacein, and hydroxytyrosol, which have antioxidant abilities [19
] and anti-inflammatory effects [20
]. However, there is no research on oleocanthal’s effect on uterine contraction and its related molecular mechanism. In this study, we aim to demonstrate the effect of extra virgin olive oil’s polyphenolic compound, oleocanthal, on reducing uterine contraction.
2. Materials and Methods
2.1. Drugs and Solutions
The drugs and solutions used in this work were as follows: dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA), oleocanthal (PhytoLab), acetonitrile, hexane, electrochemiluminescence (ECL) immunoassay (Thermo), oxytocin (Sigma-Aldrich, St. Louis, MO, USA, O3251) (Sigma-Aldrich, St. Louis, MO, USA, O6379), carbachol (Sigma-Aldrich, St. Louis, MO, USA, C4382), PGF2α (Cayman, Ann Arbor, MI, USA, 16010), acetylcholine (Sigma-Aldrich, St. Louis, MO, USA, A2661), Bay K 8644 (TOCRIS, Taiwan, 1544), potassium chloride (KCl) (SHOWA, Saitama, Japan, 1630-5150), calcium chloride dehydrate (CaCl2) (Wako, TX, USA, 038-00445), acetic acid (LAB-SCAN, Bangkok, Thailand, A8401E), β-Estradiol 3-benzoate (EB) (Sigma-Aldrich, St. Louis, MO, USA, E8515), ibuprofen (Sigma-Aldrich, St. Louis, MO, USA, I4883).
2.2. Experimental Animals
The experiment of uterine contraction used female Sprague Dawley rats (200–300 g), and the writhing test experiment used female Institute of Cancer Research (ICR) mice that were housed in a temperature-controlled room (22 ± 2 °C) with artificial illumination for 12 h, with food and water provided ad libitum. The study was approved by the Experimental Animal Care and Use Committee of Taipei Medical University (NO. LAC-2018-0210/NO. LAC-2018-0297). All animals received humane care in compliance with the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals, published by the National Science Council, Taiwan.
2.3. Extra Virgin Olive Oil Extracts Preparation
The extra virgin olive oil (LAUDEMIO, L8057) was mixed with acetonitrile (ACN) and n-hexane (HEX) at a ratio of 1:2:3. After mixing, the solution was separated into ACN and HEX layers using a separator funnel. The ACN layer extracts after liquid separation were weighted and concentrated by a vacuum condenser. The ACN layer extract was concentrated to a small amount of liquid and transferred to a vial. It was then concentrated to a paste through continued rotation for 1 h for further experiments. after the layer forming the paste, the remaining liquid was aspirated. Finally, DMSO was used to dissolve the paste.
2.4. Determination of Extra Virgin Olive Oil (EVOO) Extracts by Reverse-Phase Ultra Performance Liquid Chromatography-Photodiode Array (UPLC–PDA) Analysis
A photodiode array (PDA) detector set to 223 nm was used for the identification of compound oleocanthal (OC), based on the retention time (Rt) and ultraviolet (UV) characteristics. A BEH Shieid RP18 column (Waters Acquity, 2.1 mm × 100 mm, 1.7 μm) was used for the analysis, and the flow rate was set to 0.5 mL/min. The mobile phase consisted of solvents A: acetonitrile (ACN) and B: water with 0.1% formic acid. The gradient method was used for the quantification of the compounds of interest for 0–5 min: 100% A; 0% B. The column temperature was kept at 40 °C throughout all experiments, and the injection volume was 10 μL.
2.5. Uterine Preparations and Measurement of Uterine Contraction
Rats were sacrificed by carbon dioxide and the uterus was removed and placed in an organ bath. The tissue water bath contained Krebs’ solution (113 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2
, 18 mM NaHCO3
, 1.2 mM KH2
, 1.2 mM MgSO4
, 5.5 mM glucose, 30 mM mannitol, and pH 7.4). We removed the uterine fat and cut it into segments of equal length. The segments were placed in isolated organ baths containing Krebs’ solution at 37 °C with a 95% O2
and 5% CO2
supply. Each organ bath was equilibrated using 1 g of weight for at least 60 min. Uterine contractions were recorded with force-displacement transducers by using the LabScribe software [12
2.6. Molecular Docking Site
Molecular docking was performed by a PyMOL plug-in—NRGsuite [22
]—under the PyMOL version 1.8 environment. This included the detection of surface cavities in protein and was used as a target binding site for docking simulations. The structures of COX-2 bound with ibuprofen were downloaded from the protein data bank (PDB) database [23
], and ibuprofen was separated from COX-2 in PyMOL for further docking analysis.
2.7. Acetic Acid-Induced Writhing Test
The test was carried out using a previously described technique [24
]. Mice were treated with three different doses (28, 70, and 140 mg/kg) of extra virgin olive oil (EVOO) extracts and ibuprofen (120 mg/kg, P group) as a positive control. A total of 60 min after oral administration, writhing was induced by 0.6% acetic acid (10 mL/kg i.p., V group). Each mouse was placed in a transparent observation box, and the amount of writhing was recorded for 30 min after the acetic acid administration.
2.8. Oxytocin-Induced Writhing Test
This test was performed using the modified method described by a previous study [25
]. During the experiment, the female mice were injected intraperitoneally with 1 mg/kg body weight estradiol benzoate and administration of EVOO extracts (28, 70, and 140 mg/kg) or ibuprofen (100 mg/kg, P group) every day, except for the control group, by an oral gavage. Mice were induced to writhe by an intraperitoneal injection with 67 IU/kg oxytocin solution in DMSO at day 7, and we recorded the number of writhing responses that occurred 30 min after oxytocin injection. Uterine samples were collected for protein expression analysis.
2.9. 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Assay
We used a commercial kit (Dojindo, Japan) and followed the instructions to evaluate the antioxidant ability. The scavenging activity was detected using 100 μL 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) solution mixed with the sample in a 96-well microplate and incubated at room temperature for 30 min. The absorbance was measured at 517 nm using a VERSA Max microplate reader (Molecular Devices, San Jose, CA, USA) and using the following formula:
Scavenging activity (%) = [control − sample/control] × 100.
The IC50 DPPH values were obtained through extrapolation from regression analysis.
2.10. Lipid Peroxidation: Determination of Malondialdehyde (MDA)
The levels of malondialdehyde (MDA) in plasma followed the manufacturer’s instructions (TBARS Assay Kit Cayman, USA). Results were measured on a 532 nm plate using a VERSA Max microplate reader (Molecular Devices, San Jose, CA, USA).
2.11. Protein Preparation and Western Blot Analysis
To investigate whether OC affects the expression of PGF2α receptor (FP) protein in the uterus ex vivo, OC was added 10 min after the stimulation of the uterus with PGF2α, and the uterus was taken at 10, 30, and 60 min after the addition of OC for analysis.
Samples were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer containing protease and phosphatase inhibitors (Roche, Mannheim, Baden-Württemberg, Germany). Protein was quantitated by the bicinchoninic acid assay (BCA) and then resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to a polyvinylidene fluoride (PVDF) membrane, 5% bovine serum albumin (BSA) solution was used to block the empty space of the membrane. Subsequently, membranes were incubated with primary antibodies—PGF2α receptor (FP) (Cayman, 101802), COX-2 (Cayman, 160106), oxytocin receptor (OTR) (Santa Cruz, CA, USA, sc-8109), p-ERK (Cell Signaling, 9101), ERK (Cell Signaling, 9102), p-MLC20 (Cell Signaling, 3675), MLC20 (Santa Cruz, CA, USAsc-28329), Protein kinase C (PKC-δ) (BD Bio, 610397), α-actin (Santa Cruz, CA, USA, sc-32251)—at 4 °C overnight and then with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000) for 1 to 2 h. The visual signal was captured by an image analysis system (UVP BioChemi, Analytik Jena US, Upland, CA, USA). The band densities were determined as arbitrary absorption units using the Image-J software program version 1.52 t (NIH, Bethesda, MD, USA). The expression level of these target proteins was analyzed by three individual experiments.
2.12. Statistical Analysis
All data are presented as the mean ± standard error of the mean (SEM). The statistical significance of differences between the groups was analyzed by using Graphpad Prism (version 6.0). The statistically significant difference from the respective controls for each experiment was determined using a one-way analysis of variance (ANOVA) for all groups. A Student’s unpaired t-test was used for comparison between two groups. A value of p < 0.05 was considered to be statistically significant.
Dysmenorrhea has been reported to cause an increase in the production of prostaglandins, including PGF2α and of prostaglandin E2 (PGE2
), which may cause the contraction of the myometrium. In addition, studies have reported higher levels of PGF2α in patients with primary dysmenorrhea [28
]. An increase of COX-2 can increase the PGF2α in dysmenorrhea [29
]. Meanwhile, during a contraction, the calcium influx will activate the phosphorylation of myosin light chain 20 (MLC-20) to control the relaxation or contraction of smooth muscle [12
]. In an oxytocin-induced writhing animal model, the OTR can significantly increase COX-2 expression to increase the uterine contractions [30
]. The increase of COX-2 may be induced by oxytocin-activated PKC and the phosphorylation ERK signaling pathway [31
]. In uterine smooth muscle, oxytocin binds with the G protein-coupled receptor (GPCR) and produces calcium influx and diacylglycerol (DAG), which activates protein kinase C (PKC). PKC can activate several signaling pathways, such as extracellular signal-regulated kinase (ERK), to stimulate PGF2α synthesis [32
In the search for a natural compound that has NSAID-like effects, some compounds have been discovered. In vivo and in vitro studies have shown that resveratrol can significantly reduce uterine contractions [12
]. Moreover, in traditional Chinese medicine, adlay hull extracts also showed an effect on the inhibition of uterine contraction [13
]. This shows that polyphenols may play an important role in reducing uterine contraction; furthermore, they can reduce the symptoms of dysmenorrhea. Extra virgin olive oil is produced in all countries in the Mediterranean region, including Spain, Italy, and Greece [18
]. Previous studies have shown that high levels of extra virgin olive oil can inhibit platelet aggregation, lower cholesterol, and blood pressure, prevent cardiovascular disease and Alzheimer’s disease, and reduce arthritis, colon cancer, breast cancer, and prostate cancer risks [33
]. The phenolic compound extract from extra virgin olive oil contains oleocanthal, which has been proven to have anti-inflammatory and anti-oxidant effects [20
]. Oleocanthal is one of the compounds in EVOO causing irritation to the throat. Extra virgin olive oil produced in different regions has different oleocanthal concentrations, and extra virgin olive oil produced in Italy contains the highest concentration of oleocanthal (up to 191.8 ± 2.7 mg/kg). Laudemio’s extra virgin olive oil from Italy is abundant in oleocanthal content. Oleocanthal has an analgesic effect similar to ibuprofen, and long-term consumption may help prevent diseases. If the daily intake of 50 mg of extra virgin olive oil contains about 200 μg/mL oleocanthal (absorption rate is 60~90%), it can be taken up to 9 mg/day, which is equivalent to 10% of ibuprofen in an adult’s dose. However, the structure of oleocanthal is different from ibuprofen because of the analgesic effect of oleocanthal, which is also called “natural ibuprofen” [17
]. The oral irritant, in a similar manner to ibuprofen, can nonspecifically modulate TRPA1 channel activation [36
]. The expression of TRPA1 is strongly correlated with dysmenorrhea severity in endometriosis patients [37
], and the activation of TRPA1 may trigger the calcium influx, while EVOO may decrease TRPA1 expression and calcium influx in Alzheimer’s disease [38
This study is the first to demonstrate that extra virgin olive oil extracts and its active ingredient, oleocanthal, suppress PGF2α-induced uterine contractions in sprague dawley (SD) rats and can effectively inhibit pain in ICR mice. An EVOO ACN layer extract inhibited PGF2α induced uterine contractions in an ex vivo study; then, its active ingredient oleocanthal inhibited PGF2α, oxytocin, Ach, and carbachol-induced uterine contractions in rats. Oleocanthal also inhibited Bay K 8644, which is the calcium channel activator, and high K+ (KCl)-induced uterine contractions. In addition, oleocanthal can block PGF2α receptor to block Ca2+ influx through voltage-operated Ca2+ channels (VOCs). Finally, EVOO ACN layer extracts can effectively suppress pain in systemic pain or dysmenorrhea models in mice.
When PGF2α, oxytocin, carbachol, and acetylcholine are present, they will bind to an intracellular Gq-protein receptor and allow extracellular calcium to enter the cell via the calcium channel. When the cells are in a high potassium (>30 mM) environment, the VOC (L-type Ca2+
channel) will be turned on, allowing extracellular calcium to flow into the cells [39
]. Our current study demonstrates that OC inhibits PGF2α, oxytocin, carbachol, or acetylcholine-induced uterine contractions, as well as Bay K 8644 or KCl-induced uterine contraction, possibly by reducing the calcium ion concentration and blocking calcium influx.
The main symptom of dysmenorrhea is lower abdominal pain. In order to simulate pain response, in the current study, we used acetic acid or oxytocin to induce a writhing reaction in ICR mice. The occurrence of writhing represents pain. The writhing reaction is characterized by abdominal wall contraction, pelvic rotation, and hind limb extension [40
]. The acetic acid-induced writhing test is widely used to evaluate anti-inflammatory and analgesic activities. It increases the concentration of PGF2α and PGE2
in the peritoneal fluid and stimulates vasoconstriction, increasing the sensitivity of the peripheral nerves to prostaglandins [41
]. In osteoarthritis, lipid peroxidation increases COX-2 and causes pain [7
]. The antioxidant ability of OC reduces oxidative stress and lipid peroxidation, which reduces COX-2 protein expression and thus the pain. Oxytocin-induced writhing is the primary dysmenorrhea model; the uterus experiences ischemia, increasing the sensitivity of the distal nerve of the uterus to prostaglandins and then triggering the occurrence of a writhing reaction [31
]. In this study, we confirmed that EVOO ACN layer extracts (28, 70, 140 mg/kg) effectively inhibit the writhing reaction induced by oxytocin compared with the control group. Furthermore, OC was shown to have an antioxidant ability, which reduced the COX-2 expression, decreased the oxytocin-induced related signaling pathway, and reduced the TRPA1 expression to decrease the calcium influx.