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INTRODUCTION
Several statistical data highlight the insufficient target effect and efficiency of market-available painkilling drugs. Besides this, the side effects of these medications sometimes surpass the relief they provide, maintaining the relevance of the pain treatment issue. Valuable raw materials for various medicines continue to come from different active ingredients of plant and animal origin. Venom from various sources, including certain snakes, arthropods, snails, and other animals, as well as derivatives from plants like essential oils and extracts, are already utilized as analgesic and anti-inflammatory remedies.
Specifically, cobrotoxins are well known blockers of the cholinergic receptors and neuromuscular transmission [1], mamba venom blocks acid-sensing ion channels [2], and viper venoms may desensitize afferent neuron fibers and block nociceptive impulses [3]. In a previous study [4], various doses of Macrovipera lebetina obtusa (MLO) venom were investigated, starting from LD50 and at serial dilutions for intraplantar (IPL) and intraperitoneal (IP) (1.0 and 1/5, 1/10, 1/20, and 1/30 of LD50) administration, and it was demonstrated that the venom of the blunt-nosed viper (MLO) exhibited an analgesic effect (64%) at a low dose (1/20LD50). The mechanism behind this low-dose anti-pain action is not yet fully elucidated, but it may involve the modulation of certain voltage-gated channels of afferent neuron fibers responsible for transducing nociceptive signals.
Several plants, including Papaver somniferum, Cannabis indica, Erythroxylum coca, and others, display potent analgesic action. Essential oils from Lamiaceae plants, like Origanum vulgare L. (OV), have a broad spectrum of properties, including antibacterial, anticancer, analgesic, and anti-inflammatory actions. OV contains essential oil with more than 120 components, with sesquiterpene beta-caryophyllene (BCP) being one of them, exhibiting analgesic effects in both animals and humans.
Oregano essential oil from different origins, such as Italy, Greece, Turkey, and Armenia, belongs to different chemotypes [5]. OV from the Armenian highlands is classified as the fourth chemotype, characterized by a low content of carvacrol or thymol and a high content of BCP and its oxide. Despite having weak antimicrobial and anticancer properties, this essential oil demonstrates high analgesic activity [6,7].
The contemporary trend in scientific research focused on developing new drugs involves the exploration of combined preparations [8]. These formulations include multiple active ingredients, aiming to achieve the simultaneous activation of several mechanisms of the drug's effect. This study aimed to investigate the analgesic and anti-inflammation effects of a combined preparation of MLO venom and OV essential oil (OVEO) in mice and the mechanisms of its joint action.
MATERIALS AND METHODS
1. Animals
Male outbred albino mice (20 ± 2 g) were used throughout the experiments. Mice and rats were obtained from the nursery of the L. A. Orbeli Institute of Physiology of National Academy of Sciences of the Republic of Armenia (OIPH). The study was conducted according to the “Principles of Laboratory Animal Care” and was carried out following the European Communities Council Directive of September 22, 2010 (2010/63/EU) and was approved by the Institutional Review Board of the OIPH (protocol code N4, date of approval: 22.07.2021). A total of 162 animals were used.
2. Reagents
λ-Carrageenan (carrageenan), naloxone, SR144528, dimethyl sulfoxide (DMSO), and Tween 80 were purchased from Sigma-Aldrich. Diclofenac ointment 1% (Belarusian Medical Preparations Factory), sodium metamizole (Analgin®; Yerevan Chemical-Pharmaceutical Firm), diclofenac sodium (Diclofenac®; Hemofarm A.D., Vršac, Serbia) and morphine hydrochloride (Morphine, FSUE “Moscow Endocrine Plant”), were used as standard analgesic and anti-inflammatory drugs. All other solvents and reagents for extraction and ointment preparation were of commercial grade.
3. MLO venom obtaining, qualitative and quantitative analysis
MLO venom was milked in the serpentarium of OIPH and freeze-dried (Biobase, BK-FD10S). The mentioned venom content is described in detail in the previous study [9]. Enzymatic activity for phospholipase A2 (PLA2) and metalloproteinases was determined as in [10]. The same method was used to determine PLA2 activity in combined preparation.
5. Preparation of injectable solutions and ointment
To provide researchers with hormesis doses of venoms, the toxicity of the MLO venom was previously determined. The average LD50 value for the IP administration of the MLO venom was 1.85 mg/kg body weight for mice according to the Behrens method and 1.74 ± 0.2 mg/kg body weight according to the Miller-Tainter method [12,13]. The present investigation used 1.8 mg/kg as the mean value of LD50 of MLO venom (IP). The most effective analgesic doses for the separately used compounds (MLO venom and OVEO, IP injection) of the tested preparation were established earlier [4,7]. So each mouse was injected with the solution aliquot of 0.1 mL, where doses of active compounds in the combined preparation were 1.8 μg of MLO venom (taking into account that LD50 = 1.8 mg/kg or 36 μg per mouse for IP injection, so 1/20 LD50 = 1.8 μg), and 4.0 μL of OVEO (4% of OVEO in 0.1 mL = 4.0 μL or about 3.5 mg, according OVEO density). The saline solution of combined preparation contained 1% DMSO and 2% Tween 80.
Lanolin was used as the base for the ointment. The active ingredients of the ointment—MLO venom and OVEO—were added to the ointment base one day before the experiments. The ointment composition and the preparation process were patented (patent code number: 839Y). The ointment was stored in a refrigerator in a tightly closed glass container. The single treatment amount of the ointment for the hind paw of the mice was 40 mg. The quantity of MLO venom was the same as for IP injection, 1.8 μg for treatment, to exclude toxic effects. The OVEO dose was decreased (3.2 μL per treatment), taking into account that in the case of transdermal application, the local action of the essential oil is more strongly expressed and its systemic degradation is delayed. Separately, MLO-ointment, which contains only MLO venom, and OVEO-ointment, which contains only OVEO, were also investigated.
Hereafter in the text of the present study, ‘MLO + OVEO’ indicates a liquid solution for IP injection, and ‘ointment’ means MLO + OVEO in the lanolin-based preparation for skin treatment.
6. The formalin induced nociception assessment
The antinociceptive properties of the combined liquid preparation and ointment were assessed in mice through the formalin test (FT) [14]. Nociceptive behavior was assessed as described [7].
In the FT, the biphasic nociceptive reaction involves distinct contributions from different types of sensory nerve fibers, primarily C fibers and Aδ fibers, which mediate the phases of pain.
Phase 1 is mediating by Aδ fibers and C fibers. Aδ fibers are activated first, due to the direct chemical irritation caused by formalin, resulting in an initial sharp pain. C fibers are also activated directly, sustaining the acute pain sensation through their slower conduction. This phase is primarily peripheral and reflects the immediate excitation of nociceptors by formalin. Phase 2 is considered an inflammatory pain phase. Predominantly C fibers are involved in transmitting pain signals during the prolonged inflammatory phase. This phase is driven by an inflammatory response initiated by formalin-induced tissue injury. Pro-inflammatory mediators sensitize C fibers, leading to persistent activation. This division by fiber type highlights how the test provides insights into both acute nociceptive pain and persistent inflammatory pain, which is critical for evaluating analgesic mechanisms [15].
The selection of dosages for standard medications was guided by widely used protocols in the literature [16]. To evaluate the antinociceptive effect of the ointment, on the plantar surface of the right hind paw, 40 mg of ointment was applied and gently rubbed 50 times with the index finger of the researcher [17]. MLO-ointment and OVEO-ointment also were tested under the same conditions.
Ointment base and 1% diclofenac ointment were used as the control and reference drug, respectively. Thirty minutes later, 0.02 ml of 5% formalin was administrated by an IPL injection into the right hind paw of the mice.
7. Carrageenan-induced inflammation assessment
The carrageenan-induced edema in a mouse's hind paw was carried out [18]. The carrageenan 1% solution in saline was freshly prepared immediately before the experiments. Edema was induced by an IPL injection of 50 µL carrageenan solution into the paw of each animal in all groups, which resulted in severe swelling that reached maximum volume in 2–4 hours and decreased after 24 hours. In the positive control group, the mice were injected with 0.1 mL diclofenac solution (10 mg/kg or 200 μg/mouse, IP) immediately after the carrageenan IPL injection. The experimental animals were injected with 0.1 mL of combined preparation, IP).
In case of ointment testing, in the positive control group, the paws of the animals were treated with 40 mg diclofenac 1% ointment immediately before the carrageenan IPL injection. The experimental animals were treated with the same amount of testing ointments (MLO-ointment, OVEO-ointment, and MLO + OVEO-ointment). The edema volume was measured using a digital Plethysmometer (Ptm-Xn, Milton Enterprises) after 1, 2, 4, and 24 hours of carrageenan injection.
8. The hot plate test
The hot plate test provides cutaneous pain sensitivity latency measurements for thermal impact [19]. Acute thermal pain was evaluated by conducting the hot plate test [20]. All tested solutions were injected IP (0.1 mL), and ointments were applied to the hind paw pad 15 and 30 minutes before the mice were transferred to the hot plate (55oC ± 1oC). The experimental procedure is described in the previous study [7]. If there was not any behavioral response for 25 seconds, animals were evacuated from hot plate to avoid any paw skin damage. In repeated measurement on the hot plate test, mice are likely to learn to adapt their behavior to avoid discomfort [21], so all experimental mice were used only once.
9. Involvement of opioid receptors (ORs) in the peripheral antinociceptive effect of the ointment
To determine the role of ORs in the antinociceptive effect of the ointment, a non-selective opioid antagonist, naloxone, was used. Naloxone (20 µg/20 μL, IPL) was injected 30 minutes before the formalin IPL injection [22].
10. Involvement of cannabinoid CB2 receptors in the peripheral antinociceptive effect of the ointment
To determine the participation of CB2 receptors in the antinociceptive effect of the ointment, a selective CB2 receptor reverse agonist SR144528 was used. SR144528 (20 μg/20 μL, IPL) was injected 30 minutes before the formalin IPL injection [23].
11. Skin irritation test
The control and experimental group (4 mice in each) were chosen. The hair of the animal’s back was gently shaved. For a week, once a day, 50 mg of cream was applied to 4 cm2 of skin area. The treated area was covered with a cotton bandage, and any sensitivities were assessed and graded (A. No edema, B. Slight erythema, C. Up to severe edema, D. Moderate erythema, E. Severe erythema) [17].
12. Determination of the toxicity of the combined preparation
The experiments were conducted on 5 groups of mice (6 mice in each group) to determine the toxicity of the 1/20LD50 MLO + 4% OVEO mixture. The therapeutic dose, and 2×, 3×, 4×, and 5× times higher than therapeutic doses, were injected (0.1 mL, IP). The number of dead animals was recorded after 24 hours and the percentage of mortality in each group and LD50 were calculated using the Behrens and Miller-Tainter methods [24].
13. Statistical analyses
All statistical analyses were done using Graph Pad Prism 8.0.1 software (Graph Pad Software Inc.) were carried out. A P value of less than 0.05 was considered significant (not significant (ns) - P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). The results represent mean ± standard error of mean.
RESULTS
1. The determination of optimal doses of components for a combined preparation
The study of fresh batches of MLO venom and OVEO showed full compliance with the venom's enzymatic activity and the essential oil's gas chromatography–mass spectrometry data with the previous ones. So, in the present study, the choice of doses of active components for the combined preparations was based on data on their optimal doses, which were previously determined separately for MLO venom [4], and OVEO [7]. The testing of activity of PLA2 of the MLO venom in the combined preparation showed the presence of enzymatic activity during long-term incubation with OVEO.
2. Effects of the MLO + OVEO combined solution
1) Effect of the combined solution on formalin-induced pain
The results obtained in the FT showed that the analgesic action of the combined preparation with two active components, Macrovipera lebetina obtusa venom and essential oil of Origanum vulgare, gains its maximal intensity at 30 minutes after its injection (Fig. 1). Pretreatment with the combined preparation was performed 15 minutes, 30 minutes and 45 minutes before formalin injection. Summarized data for 45 minutes didn’t show significant pain relief due to high sensitization in the initial phase.
According to the authors' previous studies [7], the highest therapeutic activity of standard analgesics is observed when they are injected IP 15 minutes before formalin IPL injection. Fig. 2 compares the most effective action of all medications related to the duration of exposure. After the time of expressed analgesic action of preparation was determined (30 minutes), its effectiveness was compared to widely-used anti-pain and anti-inflammatory standards—analgin (166 μg/mouse in 0.1 mL, IP), diclofenac (200 μg/mouse in 0.1 mL, IP) and morphine (200 μg/mouse in 0.1 mL, IP).
In the second phase of the FT, the preparation worked more effectively than analgin and was equivalent to the effect of diclofenac (Fig. 2).
2) Effect of the combined preparation on carrageenan-induced inflammation
The anti-inflammatory effects of the combined liquid preparations were evaluated using the carrageenan test. The obtained results were compared with the effect of the standard anti-inflammatory drug diclofenac. The systemic distribution of combined preparation did not exert a statistically significant effect on edema volume, except after 2 hours of carrageenan injection, which is the most manifested time for edema formation (Fig. 3).
As can by seen from Fig. 3, there was no effect after 1 hour of injection or during long-lasting observation up to 24 hours. A significant effect was observed after 2 hours of edema formation. Diclofenac was more effective as an anti-inflammatory drug.
3) Effect of combined preparation on the hot plate test
The possible mechanisms of involvement of thermosensitive receptors in the antinociceptive action of the combined preparation were studied using the hot plate test (Fig. 4). The data obtained did not show significant changes related to thermosensitive activity both for the tested preparation and used standard pain relievers.
4) Toxicological study of the combined preparation
The toxicological studies showed that the double therapeutic dose of the combined preparation caused 40% mortality. The therapeutic range was very narrow (Fig. 5).
As was mentioned earlier, the combination of 1.8 μg of MLO venom and 4.0 μL of OVEO (1/20 LD50 MLO + 4% OVEO mixture) was chosen as the therapeutic dose. But in higher doses, MLO + OVEO exerted increased toxic effect in doses about 10-fold less, than MLO venom alone: 0.22 mg/kg versus 1.86 mg/kg for MLO (for mice). The toxicity data were proved by behavioral discomfort in the mice, which was manifested in decreased motion and some postures, which are characteristic for abdominal pain (acetic acid writhing-like behavior). So, OVEO extremely increases MLO toxic action.
Therefore, the ointment formulation of the combined preparation was used for the subsequent experiment.
3. Effects of MLO + OVEO combined ointment
1) Effect of combined ointment on formalin-induced pain
The analgesic effect of the ointment was compared with the effects of MLO-ointment, OVEO-ointment and 1% diclofenac ointment (40 mg/paw) (pretreatment time: 30 minutes). In the second phase of pain development, the ointment showed 68.2% pain relief, while the pain relief of diclofenac was 36.0% (Fig. 6).
2) Effect of the ointment on carrageenan-induced inflammation
The ointment showed an effective anti-inflammatory effect, reducing the volume of inflammatory edema by 29.0%, 36.0%, 28.0%, and 31.4% for 1 hour, 2 hours, 4 hours, and 24 hours respectively, after carrageenan (1%, 50 µL, IPL) injection. For diclofenac used as the standard anti-inflammatory drug, the efficacy of edema volume reduction was 21.0%, 12.0%, 13.3%, and 14.2% respectively (Fig. 7).
Values for all four tested ointments were compared to the inflammatory response to carrageenan at 1, 2, 4, and 24 hours, with the carrageenan response set as 100%. The complete data is shown in Table 1.
All tested ointments, including diclofenac, have shown a decrease in the volume of carrageenan-induced edema. The maximal decrease was noted in the case of the combined ointment 2 hours after treatment. Over 24 hours, the combined ointment exerts the best result compared to OVEO alone and MLO alone ointments. Considering that during the simulation of inflammation induced by carrageenan, the maximum volume of edema was recorded 2 hours after the carrageenan injection [16], the anti-inflammatory effect of the combined preparation is quite effective at this stage. Still, the effect of the ointment lasts longer than that of the diclofenac.
3) Investigation of involvement of CB2 and ORs in the tested ointment’s antinociceptive action
To identify the involvement of cannabinoid receptors, the selective CB2 receptor inverse agonist (antagonist) SR144528 was used. The injection of SR144528 (20 μg/paw, IPL) decreased the local antinociceptive effects of the ointment in the FT second phase (73%) (Fig. 8).
To reveal the involvement of the ORs, naloxone was used. The injection of naloxone (20 μg/paw, IPL) decreased the local antinociceptive effect of the ointment in the second phase (64%) (Fig. 9).
To estimate the effectiveness of the tested preparations and ointments, the areas under curve for the second phase of the FT curves were calculated (Table 2).
4) Skin irritation test
The result showed (Fig. 10) that the ointment did not induce any allergic symptoms such as inflammation or irritation in mice up to seven days after application.
DISCUSSION
As described above, the authors have developed two forms of a new preparation: an injection solution and an ointment. The analgesic and anti-inflammatory effects studies of both forms have shown promising results. Toxicological studies of the liquid combined preparation, in case of IP injection, have shown that the range between the toxic and the therapeutic doses is quite narrow, so the ointment version of the combined preparation was chosen as a new, effective analgesic and anti-inflammatory remedy.
Upon IP injection, viper venom components (e.g., proteins, enzymes, peptides) are absorbed into the peritoneal cavity and enter systemic circulation via the extensive capillary network of the peritoneum. The venom contains enzymes like PLA2, metalloproteinases, and serine proteases, which can interact with local tissues, causing inflammation and cytotoxicity. The smaller amount of venom results in reduced local damage, potentially slowing absorption as tissue inflammation and vascular permeability are less pronounced compared to higher doses. Venom proteins and peptides bind to plasma proteins and are distributed throughout the body. Specific components of the venom preferentially target endothelial cells leading to mild endothelial disruption, platelets, and clotting factors, and may trigger subclinical coagulation disturbances. Low-dose neurotoxins may cause subtle changes in nerve signaling without overt paralysis, as the venom quantity is insufficient to overwhelm physiological detoxification pathways. At low doses, many venom components may not reach the threshold needed to activate their specific toxic effects.
The analgesic effect of low doses of viper venom may seem paradoxical, given the pain-inducing properties of venom at higher doses. However, there are plausible mechanisms, supported by scientific observations, that explain how low doses of viper venom could exert an analgesic effect: activation of endogenous pain inhibition pathways such as endorphin release or descending pain modulation, desensitization of nociceptors such as TRPV1, and Nav channels. In addition, peripheral adaptation may develop, such as receptor downregulation, as well as local neurotoxicity at low levels of venom doses. Venom-induced hormesis can be manifested as activation of protective or adaptive mechanisms that suppress nociception or inflammation [25]. In cases of cytokine modulation, venom can influence the production of cytokines. At low doses, it might stimulate the release of anti-inflammatory cytokines (e.g., interleukin-10) or inhibit pro-inflammatory cytokines (e.g., tumor necrosis factor-α), contributing to analgesia.
So, there is sufficient information about the analgesic effect of viper venom PLA2 both in the scientific literature and the authors’ studies [4,26,27]. Although the specific mechanism of action of blunt-nosed viper’s venom has not been shown at the molecular level yet, the most likely mechanism is desensitization of the sensory endings of pain receptors when different voltage-gated channels of the neuronal membrane are modified by the PLAA2 of the venom. This effect has been shown e.g., for the PLA2 from the viperid venom (Agkistrodon blomhoffii ussurensis), which contains Gln49 in its active center [3]. Today, different modifications and isoforms of venom phospholipases A2 have been identified, which contain different amino acid residues in the active center, including Asp49, Lys49, Gln49, and some others. PLA2 containing Lys49 is considered to lack enzymatic activity, and Asp49 (which is characteristically proved for a lot of vipers’ venoms and particularly for MLO [9]) has pronounced enzymatic activity. Moreover, many isoforms of phospholipases, in addition to enzymatic activity, exhibit activity as toxins and signaling molecules. Particularly Gln49-PLA2, very similar to Asp49-PLA2, markedly affects the function of voltage-gated ion channels and blocks neuronal signal transduction in the nerve terminal. This is the main mechanism of analgesic action in Gln49-PLA2 [3]. The analgesic effect of MLO venom is also thought to be related to increased activity of sodium and the blockade of potassium voltage-gated channels.
At low doses of venom, when the synthesis of prostaglandins is limited due to small amounts of arachidonic acid release, prostaglandin receptors may lead to the activation of anti-inflammatory processes [28]. This fact has been described for prostaglandin D2 [29]. In addition, low amounts of PLA2 can be rapidly bound by the trans-membrane glycoprotein and undergo internalization. Then phospholipase inactivation and cleavage in lysosomes take place. Low doses of venom can lead to marked desensitization of afferent pain nerves without significant inflammatory processes. The authors have shown that it is the enzymatic activity of Asp49-PLA2 that is responsible for the analgesic effect of viper venom, used in small doses (about 1/20 LD50, which is about 90 µg/kg body weight for mice when administered IP) [4]. However, with other routes of administration—intravenous, intramuscular, or for example, subcutaneous—effective analgesic doses vary greatly. When viper venom, as part of a classic lanolin ointment, is spread on the skin in the area of inflammation, its components are deposited in the subcutaneous fatty tissue and its effect is delayed compared to IP administration.
Another active component of the combined preparation, OVEO, has a practically proven analgesic and anti-inflammatory effect [6,7]. Gas chromatographic analysis and mass spectrometry showed that OVEO contains more than a hundred different low-molecular substances (flavonoids, sesquiterpenes, and others) [7,30].
Upon IP injection, BCP, a bioactive sesquiterpene, is absorbed through the peritoneal cavity’s capillary network into the bloodstream. Its lipophilic nature facilitates rapid absorption into lipid-rich tissues and blood plasma. BCP is transported via the bloodstream and widely distributed due to its high lipophilicity. Even inhaled, BCP preferentially accumulates in lipid-rich tissues such as the liver, adipose tissue, and brain [31]. It crosses biological membranes, including the blood-brain barrier, potentially interacting with the endocannabinoid system by binding to CB2 receptors. BCP binds to plasma proteins, which modulates its bioavailability and distribution. The metabolism produces oxidized derivatives, such as epoxides (e.g., caryophyllene oxide, a major metabolite) and hydroxylated products that enhance water solubility. BCP activates CB2 receptors, reducing inflammation and oxidative stress. This action might influence its metabolism by reducing inflammatory-mediated metabolic shifts. BCP and its metabolites can reduce oxidative stress in tissues, potentially modifying local metabolic pathways. In some tissues (e.g., brain), BCP may undergo local enzymatic transformations contributing to neuroprotective effects.
In the case of the ointment, BCP acts as an analgesic agent, which is an agonist of cannabinoid CB2 receptors, the activation of which leads to the effect on keratinocytes and the subsequent synthesis and release of endorphins, which reduces local acute and inflammatory pain [6]. Undoubtedly, depending on the dose, the effect of essential oil can be irritating (high dose), or soothing (low dose). Unlike venom, which includes high molecular weight components, such as polypeptides with a molecular weight of 3–4 kDa and proteins with a molecular weight from 13 to 70 or more kDa, essential oil components are low molecular weight substances with an order of 120–250 Da. Moreover, all components of the venom are hydrophilic substances, very soluble in aqueous solutions, while the components of the essential oil have exclusively lipophilic properties. Therefore, oregano essential oil applied to the skin is absorbed more quickly and reaches its targets. For example, BCP, after absorption, is almost completely distributed in all organs, passing all histo-hematic barriers, including the blood-brain barrier [32]. In other words, the two active components in the combined preparation have fundamentally different methods of action, depending on their absorption and distribution paths in the body.
The intensity and duration of the analgesic and anti-inflammatory effect of the preparation may exceed the same abilities of individual raw materials. That is, after spreading the combined ointment on the skin, the essential oil components are absorbed first, and the venom components are deposited in the subcutaneous tissue. Of particular interest is the MLO venom, which demonstrate significant anti-inflammatory effect in the carrageenan test when used alone. However, its antinociceptive action in the FT test is considerably delayed due to its poor permeability across the skin (Figs. 6, 7). The combination of MLO venom with OVEO, which is a proven penetration enhancer (Fig. 11), assumes an increase of permeability of such hydrophilic macromolecules, such as enzymes and polypeptides of venom. So, the OVEO plays a dual role in the ointment, exerting antinociceptive properties and helping the venom to more easily penetrate the multiple layers of the skin.
The β-caryophyllene of the OVEO binds to CB2 receptors, leading to the activation of keratinocytes and the release of endorphins, leading to the activation of ORs and an analgesic effect [6]. The comparative investigation of Ointment + Naloxone and Ointment + SR144528 shows that the CB2R participation in the analgesic effect is more accented than that of the ORs. The blockade of the CB2Rs by SR144528 showed that their participation in the ointment's analgesic mechanism was much more significant than that of ORs blocked by naloxone.
Along with the activation of the ‘exoCBs - CB2R - keratinocytes - opioid release – OR’ cascade, the components of the venom manage to leave the epidermal strata, reaching the afferent nerve endings and causing their significant desensitization and blocking of nociceptive signal transduction. In addition to β-caryophyllene (8.2%), OVEO contains β-caryophyllene oxide (13.4%) [7]. There is some evidence that the β-caryophyllene oxide may contribute its anti-inflammatory and related analgesic action in tested ointment effect [33,34]. So, the OVEO action is combined, and its components may exert an “entourage” effect. The special interest may concern other components of essential oil and venom, which can provoke some hypersensitization in cases of experimental blockade of the CB2Rs, as can be seen in Fig. 8 and Table 2. The most detailed molecular mechanisms of the effect of the ointment have not yet been revealed, but it is a fact that the ointment reduces inflammatory pain by more than 68% and reduces the volume of inflammatory edema up to 36%. At the same time, according to the research data, it does not irritate the skin or have any allergic effect.
Also, there is not any data suggesting that viper venom has any addictive properties, and for OVEO, moreover, there is some evidence that it inhibits methamphetamine-taking and methamphetamine-seeking behaviors in rats [35].
So, the authors tested the recipe for a new analgesic and anti-inflammatory preparation that does not contain any compounds that can cause addiction. Due to the content of its two main components, tested in optimal proportions, this preparation has a dual action: desensitization of nociceptive nerve fibers with a decrease of nociceptive impulses transduction (venom), and activation of peripheral opioid and cannabinoid anti-nociceptive system with a decrease of nociperception in central structures of the brain (essential oil). So, the tested combined preparation may be a candidate for the development of prospective anti-nociceptive and anti-inflammatory drug, but there is a need for further investigation of this preparation’s dermatokinetics and the exact mechanisms of its action. Despite the experimental data having shown that OVEO facilitates MLO venom toxicity upon IP injection, the mechanisms of the mutual action of the venom and essential oil has not been investigated yet.
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