Korean J Pain 2022; 35(2): 140-151
Published online April 1, 2022 https://doi.org/10.3344/kjp.2022.35.2.140
Copyright © The Korean Pain Society.
Armenuhi Moghrovyan1 , Lilya Parseghyan2 , Gohar Sevoyan3 , Anna Darbinyan2 , Naira Sahakyan4 , Monica Gaboyan5 , Zaruhi Karabekian3 , Armen Voskanyan2
1Department of Pharmacognosy, Yerevan State Medical University after M. Heratsi, Yerevan, Armenia
2Orbeli Institute of Physiology, Laboratory of Physiologically Active Substances Investigations, Yerevan, Armenia
3Orbeli Institute of Physiology, Laboratory of Tissue Engineering, Yerevan, Armenia
4Yerevan State University, Research Institute of Biology, Faculty of Biology, Yerevan, Armenia
5Yerevan State Medical University after M. Heratsi, Faculty of Pharmacy, Yerevan, Armenia
Correspondence to:Armen Voskanyan
Orbeli Institute of Physiology, National Academy of Sciences, 22 Orbeli Bros. str. 0028 Yerevan, Armenia
Tel: +374 94818197
Fax: +37410 2802050
Handling Editor: Jong Yeon Park
Received: November 23, 2021; Revised: February 1, 2022; Accepted: February 2, 2022
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: Essential oils are of great interest for their analgesic and anti-inflammatory properties. We aimed to study the content of the essential oil of the Origanum vulgare of the Armenian highlands (OVA) in different periods of vegetation and to investigate its antinociceptive and anti-inflammatory effects in mice (in vivo) and cytotoxic action in cultured cells (in vitro). OVA essential oil was extracted from fresh plant material by hydro-distillation.
Methods: For OVA essential oil contents determination the gas chromatography-mass spectrometry method was used. Formalin and hot plate tests and analysis of cell viability using the methyl-thiazolyl-tetrazolium (MTT) assay were used.
Results: The maximal content of β-caryophyllene and β-caryophyllene oxide in OVA essential oil was revealed in the period of blossoming (8.18% and 13.36%, correspondently). In the formalin test, 4% OVA essential oil solution (3.5 mg/mouse) exerts significant antinociceptive and anti-inflammatory effects (P = 0.003). MTT assay shows approximately 60% cytotoxicity in HeLa and Vero cells for 2.0 μL/mL OVA essential oil in media.
Conclusions: The wild oregano herb of Armenian highlands, harvested in the blossoming period, may be considered as a valuable source for developing pain-relieving preparations.
Keywords: Analgesia, Anti-Inflammatory Agents, Beta-Caryophyllene, Caryophyllene Oxide, Cell Survival, Gas Chromatography-Mass Spectrometry, Nociception, Oils, Volatile, Origanum, Pain, Pain Measurement.
Interest in the raw materials of natural herbs, already great, is increasing day by day . The flora of Armenia is rich in its variety of herbal raw materials . Plants of the
The composition of biologically active substances in plants are defined by growth, as well as climatic and other environment factors  which have great influence on the essential oil production dynamics . The content of essential oil from the
The exact mechanism of the essential oil’s influence on the endocannabinoid system activity is still poorly understood . There are two types of cannabinoid receptors. Type 1 (CB1) cannabinoid receptors are predominantly present in the central nervous system, making them a potential target for neuropsychological disorders and neurodegenerative diseases. Therefore, CB1 activation can cause psychosis and panic in some cases. On the other hand, inhibition of CB1 can lead to depressive or anxious behavior . Cannabinoid receptors of the type 2 (CB2) are widely represented in the immune system and the periphery (in leukocytes and lymphocytes, mast cells, and microglia), which determines their participation in immune modulation . CB2 is a promising target for treating inflammatory diseases, neuropathic pain, and immune modulation . CB2 activation can affect different signaling pathways. Cannabinoids are able to modulate the function of immune cells and thus influence the secretion of cytokines. Inflammation plays a decisive role in the defense of the immune system against damaging factors such as pathogen attack and mechanical injury. Exogenous as well as endogenous cannabinoids can modulate pain and inflammation both through a direct influence on appropriate receptors as well as by inhibiting inflammatory neuropeptide release .
Pain relief is a major challenge in the current health care system. Pain is probably the most common symptomatic reason to seek medical consultation. Despite improved knowledge of the underlying mechanisms and better treatments, many people who have any type of pain receive inadequate care and non-effective drugs. There is a proven causal link between pain and inflammation. In inflammation, key players are macrophages, T-lymphocytes, cytokines, and chemokines. In the spinal cord and brain, microglia and astrocytes are involved in these processes too. Many physiologically active substances reduce inflammation, resulting in pain relief .
The aim of this research was to reveal the chemical composition of OVA essential oil, and to study its possible analgesic, cytotoxic, and anti-inflammatory properties.
Male outbred albino mice (4–5 weeks old, 20 ± 2 g) were used throughout these experiments. Mice were obtained from L. A. Orbeli Institute of Physiology National Academy of Sciences of the Republic of Armenia (NAS RA). The animals were maintained in a room with a controlled temperature (22°C ± 2°C) and a 12-hour light/dark cycle, and all mice had unlimited access to food and water. Twelve hours before each experiment, the animals received only water to avoid possible interference of the food with the absorption of the drug. The study was conducted according to the “Principles of Laboratory Animal Care” and was carried out in accordance with the European Communities Council Directive of September 22, 2010 (2010/63/EU) and was approved by the Institutional Review Board of the Orbeli Institute of Physiology of NAS RA (protocol code N4, date of approval: 22.07.2021).
For the formalin test, eleven groups (six mice in each group) were investigated. During the implementation of the hot plate test, there were two groups of mice (twelve in each). Totally, ninety mice were used. No animal was euthanized during experiments.
The primary processing of the raw materials was carried out immediately after collection: discarding organic and mineral mixtures, washing, and drying .
OVA essential oil was extracted from fresh plant material (aerial parts only) by hydro-distillation for 3 hours, using a Clevenger-type apparatus. The distilled essential oil had been dehydrated with anhydrous sodium sulfate and stored at 4°C in dark, airtight bottles until further analysis .
The essential oil composition was analyzed at the FDA Analytical laboratory (Tonus-Les LLC., Yerevan, Armenia). The gas chromatography (GC) analysis was carried out using a Bruker gas chromatograph (Bruker 450-GC; Bruker Corporation, Billerica, MA), fitted with 60 m × 0.25 mm × 0.25 μm OPTIMA-FFAP column (Macherey-Nagel, Düren, Germany). The oven temperature varied from 40 to 220°C with a scanning rate of 3°C/min, evaporator temperature was 220°C. Helium (purity 5.6) was used as a carrier gas at a flow rate of 1 mL/min. The GC was equipped with a Hewlett–Packard 5972 Series mass spectrometry (MS) detector. The MS operating parameters were an ionization voltage of 70 eV and an ion source temperature of 250°C. The diluted samples of essential oils of 2 µL had been injected manually. To avoid overloading the GC column, the essential oils were diluted 1:50 (v/v) in methanol. The identification of peaks was carried out based on a library search using the NIST–2013 . It was also determined retention time and retention index would be basic parameters for the descriptive components, which are very important for the chemometrics of essential oils . The substances are presented in ascending order of retention indices (Table 1).
As a standard analgesic sodium metamizole (Analgin®; Yerevan Chemical-Pharmaceutical Firm, Yerevan, Armenia) and as a standard anti-inflammatory agent diclofenac sodium (Diclofenac®; Hemofarm A.D., Vršac, Serbia) (positive controls) were used. The formalin (Sigma-Aldrich, St. Louis, MO) and other chemicals were analytical or sequencing grade.
The formalin test was used to evaluate the antinociceptive properties of the OVA essential oil after intraperitoneal injection in mice . Counting the bites/licks of the hind paw was used for estimating nociceptive behavior. An intraplantar injection was performed on the hind paw of the mice, using 20 μL of 5% formalin. The nociceptive behavior (the biting/licking number) was recorded for 45 minutes. Sodium metamizole (Analgin®, 8.3 mg/kg) and sodium diclofenac (Diclofenac®, 10 mg/kg) were used as standard drugs. Dosage selection was based on protocols widely used in the literature . The initial mixture of OVA essential oil for the experimental groups were made up of 50 μL dimethyl sulfoxide (DMSO), 100 μL Tween 80, and 50, 100, 150, 200, and 250 μL of OVA essential oil in 1 mL saline (respectively for each group). Then, the volume of the solution was brought to 5 mL using saline. Tested solutions were injected intraperitoneally (15 minutes and 30 minutes before formalin injection), with the concentrations of OVA essential oil being 2%, 3%, 4%, and 5% per mouse, respectively. The actual amount of essential oil administered to one mouse corresponded to 2.0, 3.0, 4.0, and 5.0 μL (essential oil density is equal to 0.87, the OVA essential oil mass was 1.74, 2.60, 3.48, and 4.35 mg/mouse) according to one or another group of experimental animals. The whole experiment lasted 60 or 75 minutes from the moment the essential oil was injected. The quantity of biting/licking of the hind paw was recorded for 45 minutes after intraplantar injection of formalin.
This test was used to assess acute thermal pain . The mice (20 ± 2 g) were placed into a Plexiglas cylinder, one by one, on a surface maintained at a stable 55°C. An intraperitoneal injection of 0.1 mL 4% OVA essential oil was made in the experimental group after 15 minutes. The time latency of hind paw licking and/or shaking, or jumping, was recorded, and at this point the animal was immediately removed from the hot plate to avoid further damage and the next animal was placed on it.
The HeLa (human cervical cancer cells) and Vero cells (African green monkey’s kidney epithelial cells; American Type Culture Collection, Manassas, VA) were used. Both cell lines were cultivated in Dulbecco’s modified Eagle medium with 5% v/v fetal bovine serum (Gibco/Termo Fisher Scientefic, Rochester, MN), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified atmosphere containing 5% CO2 (Memmert ICO105). The metabolic effects of OVA essential oil were assessed by methyl-thiazolyl-tetrazolium (MTT) colorimetric assay . In all experiments both HeLa and Vero cells were seeded at 1 × 105 cells/mL (1 × 104/well of 96-well plate) a day before the MTT assay. Initial studies to test the influence of different concentrations of DMSO as a solvent of essential oil in the culture media of the HeLa cells were carried out. The 0.1% concentration of DMSO proved to be comfortable for the proliferation of both HeLa and Vero. In the first series of experiments, twenty-four hours after culture seeding, the media was replaced, and 100 μL of fresh media containing essential oil were added to each well at the following v/v ratios: 0.1, 0.25, 0.5, 1.0, and 2.0 μL/mL. The data of cytotoxicity was evaluated after twenty-four hours of essential oil addition (totally after 48 hours). In the second series of experiments, culture seeding in the media containing the essential oil was carried out at the same ratios: 0.1, 0.25, 0.5, 1.0, and 2.0 μL/mL and data of growth inhibition was evaluated after twenty-four hours.
Data analysis was performed by Graph Pad Prism 8 software (Graph Pad Software Inc., San Diego, CA). The results of the formalin test observations at each minute were averaged both for the entire experimental period (45 minutes) and in 5 minutes intervals (for the analysis of dynamic changes in nociceptive behavior). One-way analysis of variance followed by the Bonferroni multiple comparison test was used for statistical analysis. Values of
Since most plants at different stages of vegetation differ in the quality and quantity of the chemical composition of essential oils, their determination in experimental mixtures is necessary. Depending on when the plant is collected, the essential oil in one case can demonstrate pronounced antibacterial properties, in another case analgesic properties, and the third case anti-cancer effects,
The results of the quantitative and qualitative analysis of essential oil obtained in the pre-blossoming period have shown that OVA essential oil contains 20 components with concentrations of more than 1.0% (total amount 74.99%). Another 125 components were found with concentrations lower than 1.0% (total amount 25.0%) (Fig. 1).
The results of the quantitative and qualitative analysis of essential oil obtained in the blossoming period have shown that OVA essential oil contains 20 components with concentrations of more than 1.0% (total amount 80.89%). Another 120 components were found with concentrations lower than 1.0% (total amount 19.0%) (Fig. 2).
The results of the quantitative and qualitative analysis of essential oil obtained in the fruiting period have shown that OVA essential oil contains 20 components with concentrations of more than 1.0% (total amount 84.38%). Another 101 components were found with concentrations lower than 1.0% (total amount 15.6%) (Fig. 3). The GC-MS analysis of OVA essential oil additionally showed that it contains acyclic sesquiterpenes, acyclic sesquiterpene alcohols, bicyclic sesquiterpenoids, tricyclic sesquiterpenoids, tricyclic sesquiterpene alcohols, monocyclic terpenoids, bicyclic monoterpenoids, acyclic monoterpene alcohols, monocyclic monoterpene ketones, monoterpene acids, and aromatic monoterpenoids. The dominant components of OVA essential oil during different vegetation periods are shown in Table 1.
The results of the GC-MS analysis of OVA essential oil showed that it contains acyclic sesquiterpenes, acyclic sesquiterpene alcohols, bicyclic sesquiterpenoids, tricyclic sesquiterpenoids, tricyclic sesquiterpene alcohols, monocyclic terpenoids, bicyclic monoterpenoids, acyclic monoterpene alcohols, monocyclic monoterpene ketones, monoterpene acids, and aromatic monoterpenoids. As can be seen, the content of essential oil components is quite variable during different stages of growth. For the major components (with contents more than 5% in the essential oil at different periods), the data is summarized in Fig. 4. The maximal amounts of β-caryophyllene and β-caryophyllene epoxide are present in the OVA essential oil in the blossoming period. Therefore, the essential oil of
First, the formalin test for the intact mice was carried out. The analgesics were injected intraperitoneally (15 minutes or 30 minutes before the formalin intraplantar injection). The analgesic effects of Analgin and Diclofenac pretreatment 15 minutes before the formalin intraplantar injection are shown in Fig. 5.
It is known that only local anesthetics affect the acute pain phase . Both Diclofenac and Analgin showed a significant pain relief effect in the second phase (16–25 minutes,
According to obtained data, there was no significant difference between the intact and experimental groups. So, the antinociceptive effect of OVA essential oil is not related to pain and heat sensitive TRPV1 receptors. The results are shown in Fig. 10.
The essential oil of
The results of the present investigation directly confirm that the essential oil is exposed to quantitative and qualitative changes depending on vegetation period of plant (Fig. 4). The amount of the essential oil of investigated aerial parts of the oregano of the Armenian highlands changes depending on differing hours of daylight, air temperature, amount of light received, moisture, and the intensity of solar radiation. The variability of scientific data is often defined by climatic features .
The results of our investigations showed that the wild
It was shown that essential oil expresses significant antinociceptive effects for all concentrations of OVA essential oil (2%, 3%, and 4%). As was mentioned above, a 5% solution of OVA essential oil had manifested slight signs of behavioral discomfort in tested mice and we decided do not to exceed the concentration of 4%, despite the fact that it is known that, for example, β-caryophyllene is not toxic even in much higher doses . In our case, the injected effective dose of whole OVA essential oil was equal to 3.5 mg/mouse. This means approximately 286 μg/mouse in terms of β-caryophyllene (8.18%) and 467.6 μg/mouse in terms of β-caryophyllene oxide (13.36%). Based on scientific data it is known that β-caryophyllene’s effect on pain-like behavior and its anti-inflammatory action is mediated by interaction with CB2 receptors and then β-endorphin release, which lead to activation of the opioid receptors. In contrast, β-caryophyllene oxide expresses its antinociceptive and anti-inflammatory effect through interaction with central pain receptors. In addition, it is known, that both β-caryophyllene and β-caryophyllene oxide decrease the release of inflammatory mediators of pain .
The formalin test (consists of Phase 1 [0–5 minutes] and Phase 2 [15–45 minutes]) produces painful behavior in animals. These phases are separated by a quiet phase called the interphase, in which nociceptive reactions decrease or disappear completely . The formalin test allows investigation of nociceptive behavior in the time period (second phase of formalin action) when inflammatory processes and inflammation-induced pain are developing. On the basis of these data, it was chosen as the preferable test for our investigations. The results obtained in the second phase of the formalin test suggested that the antinociceptive effect of OVA essential oil is related to the inhibition of the biosynthesis of pain mediators, such as prostaglandins and prostacyclins. In the hot plate test, it seems that OVA essential oil components did not interact with heat-sensitive TRPV1 receptors, and in this case we received a null result. Presumably, OVA essential oil exerts its analgesic effect by another route, probably the CB2 receptor-activating mechanism.
There are certain articles and reviews describing the anticancer properties of β-caryophyllene and β-caryophyllene oxide [15,30]. This study has shown the cytotoxic effects of OVA essential oil on a cancer cell line
When OVA essential oil solution was added to the well after 24 hours of cultivation (with already attached cells), all investigated doses show the cytotoxic effect for cancer and non-cancer cells. When cells were seeded in media with preliminarily added OVA essential oil in the same concentration (2%), cancer cells showed a significantly higher viability (about 60%,
Armenuhi Moghrovyan: Writing/manuscript preparation; Lilya Parseghyan: Investigation; Gohar Sevoyan: Investigation; Anna Darbinyan: Investigation; Naira Sahakyan: Writing/manuscript preparation; Monica Gaboyan: Investigation; Zaruhi Karabekian: Project administration; Armen Voskanyan: Supervision.
No potential conflict of interest relevant to this article was reported.
No funding to declare.