A Eugenius Way of Anesthesia: The Effects of Different Clove Oil Anesthesia Administration Methods on Earthworm Physiology and Behavior

Abstract

Clove oil, Eugenia caryophyllata, has been implemented as home therapeutic medicine for its anesthetic effects. The active ingredient, eugenol inhibits voltage gated sodium channels, hence sedating as an anesthetic. Clove oil provides an alternative for synthetic analgesics including morphine. The typical application of clove oil consists of topical application through liquid immersion. In addition to liquid, emulsification gel application allows for another form of controlled penetrative topical drug delivery. Moreover epicutaneous application through microneedles allowed for a non-invasive administrative method of delivering clove oil between the cuticle and epidermis.  Earthworms Lumbricus terrestris were the specimens tested due to their complex neurological functionalities. Mobility, convulsions, stimuli response, regeneration, and mortality were evaluated with concentrations of 0%, 1%, 2%, 3% with gel/immersion, and additional 0.5% with epicutaneous application. Different components of the experiment were conducted within a time-span of three years. 1% epicutaneous was shown to sedate the stimuli response of earthworms at an extremely fast rate. Furthermore 1% clove oil of all administration methods were the most effective as compared to lower concentrations 0.5% and higher concentrations 2%-3%. None of the administrative methods or concentrations of clove oil application caused limitations in regeneration, convulsive movements, or increased rate in mortality.  The limitations of this experiment consist of not consistently regulating the temperature of the medium. Future implications consist of more trials for validation and evaluating vaporization as another potential application. 1% epicutaneous showed an efficient form of sedation without severe behavioral and physiological complications.

Introduction

Anesthesia

Anesthesia is utilized in modern day medicine for the purpose of protective analgesia, in a wide variety of invasive and noninvasive surgical procedures. Local and general anesthesia are common forms of treatment that are primarily used for the surgical repairment of open traumas. The complexity of anesthesia inhibition and safety are crucial factors that need to remain constant to prevent post-surgical complications. Current research suggests that certain forms of steroidal anesthesia are detrimental to the physiological characteristics of less complex species to even humans. Research on the steroid injections in the cervical spinal region of a human were demonstrated as a leading cause of neurological infarct, a neurological compilation associated with tissue necrosis [14]. Moreover, more common forms of local and general anesthesia were demonstrated to hinder factors of the morphological as well as the behavioral functionalities of less complex organisms, such as annelids. It was established that lidocaine and prilocaine, through immersion administration, causes coelomocyte extrusion and erratic behavior in earthworms [12]. Therefore, this demonstrates that anesthesia administration must be potent as well as safe when treating an organism undergoing surgical procedures. In addition to limiting the usage of certain synthetic anesthetics, offering individuals a wider selection of anesthesia would prevent complications such as drug relapse. As an example, individuals at risk for drug relapse cannot get treated with psychotropic anesthetics such as morphine [13]. Overall, the issues of certain forms of modern day local synthetic anesthetics prompt the necessity of a holistic alternative.

Clove oil

It is not one of the most broadly known herbs, however clove oil has numerous utilizations as an anticarcinogenic, antioxidant, and as an analgesic supplement. Clove oil may serve as a replacement for an anesthetic that is separated from Clove Syzygium aromaticum. Clove oil is derived from an herb that initially originates from India. Clove oil is utilized for an assortment of purposes, including as an antimicrobial, to help eliminate microscopic organisms, and as a pain reliever, to ease respiratory conditions, etc. [17]. Syzygium aromaticum contains an active chemical agent known as eugenol, which has comparable characteristics to a local anesthetic, thus making it a holistic anesthetic with antibacterial properties. Eugenol an active agent, and phenolic compound in clove oil can be compared to local anesthetics. Eugenol has the potency to inhibit voltage-gated sodium channels (VGSC), which are neurological receptors that are responsible for the communication of neurons as well as cells throughout the body and activation of transient receptor potential vanilloid subtype 1 (TRPV1), which shows sedative effects [11]. Moreover, prior studies demonstrated that low concentrations of clove oil assist in lowering the levels of stress while not altering the cardiovascular response of the aquatic organisms during transportation [5]. Furthermore, the topical application of clove oil in dentistry exemplifies another administration method of clove oil that is deemed as safe and effective [3] Since clove oil is popular in dentistry because of its soothing properties, it is available over the counter at a drugstore. There are multiple studies that suggest clove oil as a topical and local anesthetic for minor oral health issues such as toothaches. Clove oil has the potency to sedate a small portion of the mouth as a local anesthetic replacement with buccal patch serratiopeptidase [16]. Along with toothaches, cloves are alleviating the pain of dry socket, a typical circumstance that can occur from teeth extractions [3].

According to the previous experiment that was conducted regarding establishing an appropriate concentration of clove oil, it was demonstrated that the 1% concentration of clove oil was effective in sedating as a local and general anesthetic at a fast rate without causing any behavioral complications. Such results established that lower concentrations of clove oil were more effective and did not cause any neurological deficiencies. In this experiment, the concentrations of clove oil consisted of 0%, 0.5%, & 1%, based on how the 1% was demonstrated as efficient and safe.

Earthworms

The organism that was utilized for this experiment is earthworm, Lumbricus terrestris. Earthworm's bodies are composed of ring-like portions called annuli. These portions are shrouded in setae, or little fibers, which the worms use for mobility. Furthermore, earthworms are viewed as one of the most suitable specimens for ecotoxicological testing [8]. Although these annelids are one of the simpler preserved invertebrates, the digestive, circulatory, reproductive, and nervous systems are well-evolved and simple to recognize, making them ideal specimens. Specifically, the earthworms have a structure known as the cerebral ganglia that was comparable to the human brain, as they both control all the bodily functions that can range from stimuli response to regeneration [7]. Additionally, earthworms have comparable skin layers with humans because they are both used for protection and excretion [15]. Overall, there were several indistinguishable attributes, contributing to one of the main purposes of this experiment which was to evaluate the earthworm's neurological complex characteristics to establish their similarities with humans. In addition to mobility and physical behavior, Lumbricus terrestris are flexible invertebrates that have the physical ability to self-regenerate after trauma [4]. The regeneration ability of earthworms was determined by its neurological attributes, which can be affected by anesthesia. Therefore, the regeneration process and the regeneration rate were also investigated in this experiment. Regeneration was an important aspect to examine to distinguish any side effects that can possibly harm or benefit the earthworms. Additionally, regeneration represents a surgical procedure which allows the correlation between tissue healing and growth to be identified.

Immersion

Clove oil immersion is an anesthesia administration method that is comparable to topical application. Immersion consists of the process of submerging an organ or an organism in an anesthetic solution to sedate. Specifically, clove oil immersion in earthworms consists of submerging tail annuli, thus making it effective without hindering any organs such as the cerebral ganglia. Although immersion is a noninvasive form of anesthesia administration, it only sedates moderately compared to vaporization or intravenous venipuncture. Research has demonstrated that topical application of local anesthetics is noninvasive because they do not cause any histopathological changes, however, are only somewhat effective since they are only applied on mucosal and skin barriers [1]. Immersion is a noninvasive anesthesia application method but is not the most applicable for local anesthesia in more complex organisms including humans.

Epicutaneous

Epicutaneous administration is utilized in immunological, as well as dermatological medicine. This process consists of delivering a substance within the epidermis layer of an organism. Moreover, the wide variety of implications that epicutaneous administration has on delivering a substance in an organism are used to diagnose allergic reactions and to promote skin rejuvenation. In other words, the common purpose behind utilizing this application revolves around medical screening and fortifying an organism’s health. The immunological purposes consist of analyzing the severity of allergens and as a form of immunotherapy. The recognition and reduction of aero and oral allergic responses aref undamental variables that correspond to epicutaneous administration tests [2]; [10]. Moreover, forms of epicutaneous application consist of derma patching, derma stamping with microneedles (skin pricks), as well derma scratching. Research conducted on utilizing derma patching as a form of topical local anesthesia application for skin grafts as well as labor pain and was suggested as a safer alternative compared to intravenous administration [6]; [9]. Nevertheless, microneedling and derma scratching have not been utilized to apply analgesics or sedate organisms. Moreover, the dermatological implications of epicutaneous derma stamping, are extremely beneficial towards inducing skin rejuvenation from hypertrophic scarring by delivering serums through a safe and non-invasive manner. Such research indicates that epicutaneous microneedle administration is an effective drug delivery methodology that is noninvasive and efficient. Notably, administering clove oil as a form of local anesthesia through epicutaneous microneedling would indisputably sedate earthworms efficiently and noninvasively.

Nanoemulgel

Medications in the form of gel have a wide variety of benefits in terms of convenience and potency. Specifically applying medications with a gel base allows for a controlled drug delivery. Previous research on medicinal gels such as nanoemulgel shows it aids in drug penetration when applied topically. Specifically, emulsification gel allows for controlled drug delivery through the outermost layer of the epidermis skin layer [15]. Emulgel is applicable to clove oil treatment based on previous research incorporating clove oil gel for bacterial inhibition. Eugenol was previously utilized as an oil phase in the formulation of a ketoprofen nanoemulgel. It was found that the gel formulation had synergistic antibacterial activity [16]. In other words, clove oil concentrations formulated in gel can allow for an increase in anesthetic efficiency and a controlled drug release system. A form of gel, white petroleum jelly was used to formulate clove oil into a gel application. Petroleum jelly can be utilized as a moisturizing agent as well as drug delivery application. Previous research evaluated petroleum jelly as a vehicle for liposomal drug delivery through skin layers [17]. A commercialized over-the- counter form of petroleum jelly consists of Vaseline. 100% Petroleum jelly Vaseline was used as a base to establish 0%, 1%, and 2% clove oil concentrations as an emulgel.

Stimulus Response Assay

The stimulus response tests involve measuring the behavioral responses after the initial sedation of the earthworms. These tests were completed in the form of an invasive probe. Normally, hypodermic needles are used to decide how their structure impacts the simplicity of inclusion into the skin, understanding patient pain and skin injury; however, in the last experiment, a toothpick was utilized for safety purposes. Additionally, sharpness was dictated by estimating the penetration power of a probe into a known medium which intently replicates skin. A membrane of uniform thickness was held under constant strain and was repositioned to permit numerous tests to be performed [4]. Additionally, a key part of the fixturing was to guarantee that the instrument did not flex during the test and that the angle was constantly the same in order to preserve the validity. Overall stimulus response was utilized to measure the initial reaction of an organism based on physical stimuli.

Materials and Methods

Preparation of Earthworms

The earthworms, L. terrestris, were obtained from the Carolina Biological Website (25/container). The earthworms were kept in a large container with compacted mouse pellets and a Sphagnum moss medium sprinkled with dandelion root tea leaves. The earthworms were separated into bins of groups of fifteen to ensure validity and accuracy within the results. Additionally, this allowed the earthworms to be separated and protected overnight when supervision was not present. The earthworms were organized into different categories based on the type of test administration method; clove oil concentration as shown in Table 1.

Table 1. 1. Group distribution between different concentrations, and assays (2020-2021).

Type of Anesthesia Administration	Concentration	of Samples	# of Samples	Types of Assays
Immersion	0% 1.0% 2.0% 3.0%	15 Earthworms 15 Earthworms 15 Earthworms 15 Earthworms	5 Earthworms 5 Earthworms 5 Earthworms 5 Earthworms 5 Earthworms 5 Earthworms 5 Earthworms 5 Earthworms 5 Earthworms 5 Earthworms 5 Earthworms 5 Earthworms	Mobility Stimuli Response Convulsive Movements Mobility Stimuli Response Convulsive Movements Mobility Stimuli Response Convulsive Movements Mobility Stimuli Response Convulsive Movements

Table 2. Group distribution between different administration applications, concentrations, concentrations and assays (2021-2022).

Group#	Type of Anesthesia Administration	Concentration	of Samples	# of Samples	Types of Assays
Group A	Immersion	0% 0.5% 1.0%	15 Earthworms	5 Earthworms 5 Earthworms 5 Earthworms	Mobility Stimuli Response Convulsive Movements
Group B	Epicutaneous Microneedling	0% 0.5% 1.0%	15 Earthworms	5 Earthworms 5 Earthworms 5 Earthworms	Mobility Stimuli Response Convulsive Movements

Table 3. Group distribution between different administration applications, concentrations, and assays (2022-2023).

Group#	Type of Anesthesia Administration	Concentration	of Samples	# of Samples	Types of Assays
Group A	Immersion	0% 1.0% 2.0%	15 Earthworms	5 Earthworms 5 Earthworms 5 Earthworms	Mobility Stimuli Response Convulsive Movements
Group B	Nanoemulgel	0% 1.0% 2.0%	15 Earthworms	5 Earthworms 5 Earthworms 5 Earthworms	Mobility Stimuli Response Convulsive Movements

2021-2022

Figure 1. Response Time vs. Immersion Treatment. 15 earthworms in groups of 5 were immersed in concentrations of clove oil treatment ranging from 0%- 1%. The 1% concentration was the most effective in hindering the response time. Error bars represent ±SD.

Figure 2. Response Time vs. Epicutaneous Treatment. 15 earthworms in groups of 5 were immersed in concentrations of clove oil treatment ranging from 0%- 1%. The 1% concentration was the most effective in hindering response time. Based on ANOVA following a Tukey HSD test, the 1% treated group is significant compared to the control group with a *p-value<0.05. Error bars represent ±SD.

Figure 3. Response Time vs. All Treatment Groups. 15 earthworms in groups of 5 were immersed in concentrations of clove oil treatment ranging from 0%- 1%. 1% concentration of epicutaneous treatment was the most effective in hindering response time. Based on ANOVA following a Tukey HSD test, the 1% epicutaneous treated group is significant compared to the control group with a *p-value<0.05. Error bars represent ±SD.

Figure 4. Mobility vs. Time (Immersion). 15 earthworms in groups of 5 were immersed in concentrations of clove oil treatment ranging from 0% to 1%. 1% concentration of the epicutaneous treatment limited the average number of movements to under 40 movements throughout the 60-minute period. Error bars represent ±SD.

Figure 5. Mobility vs. Time (Epicutaneous). 15 earthworms in groups of 5 were immersed in concentrations of clove oil treatment ranging from 0% to 1%. There is no observable difference between the 0% and the 1% as well as 0.5% treated groups for the average number of movements per 10-minute time intervals. Error bars represent ±SD.

Figure 6. Number of Convulsive Movements vs. Time (Immersion). 15 earthworms in groups of 5 were immersed in concentrations of clove oil treatment ranging from 0% to 1%. The average number of convulsive movements stayed consistent throughout the 60-minute time interval. Error bars represent ±SD.

Figure 7. Number of Convulsive Movements vs. Time (Epicutaneous). 15 earthworms in groups of 5 were epicutaneously administered concentrations of clove oil treatment ranging from 0% to 1%. The average number of convulsive movements stayed consistent throughout the 60-minute time interval. Error bars represent ±SD.

Figure 8. Concentration vs. Average Number of Convulsive Movements. 15 earthworms in groups of 5 were immersed in concentrations of clove oil treatment ranging from 0% to 1%. 1% concentration of immersion treatment had the highest average number of convulsive movements. The average number of convulsive movements at other concentrations in both treatments remained consistent. Error bars represent ±SD.

Figure 9. Average Length vs. Days (Immersion). 15 earthworms in groups of 5 were immersed in concentrations of clove oil treatment ranging from 0% to 1%. The rates of regeneration for the 0%, 0.5%, and 1% immersion treated concentrations stayed consistent over the 4-day interval. Error bars represent ±SD.

Figure 10. Average Length vs. Days (Epicutaneous). 15 earthworms in groups of 5 were epicutaneously administered concentrations of clove oil treatment ranging from 0% to 1%. There is no observable difference between the 0%, 1%, and 0.5% treated groups for the average rate regeneration length over the 4-day interval. Error bars represent ±SD.

Figure 11. Average Mass vs. Days (Immersion). 15 earthworms in groups of 5 were immersed in concentrations of clove oil treatment ranging from 0% to 1%. The rate of regeneration at all concentrations in the immersion treatment were consistent over the 4-day interval. Error bars represent ±SD.

Figure 12. Average Mass vs. Days (Epicutaneous). 15 earthworms in groups of 5 were epicutaneously administered concentrations of clove oil treatment ranging from 0% to 1%. The rate of regeneration in the epicutaneously administered concentrations were relatively similar over the 4-day interval. Error bars represent ±SD.

2022-2023

Trial 1

Figure 13. Immersion Concentration vs. Stimuli Response (Trial 1). 15 earthworms in groups of 5 were imeeersed in concentrations of clove oil treatment ranging from 0%, 1%, to 2%. The 1% concentration of clove oil immersion was the most effective in sedating at a fast rate. Based on ANOVA following a Tukey HSD test, the 1% treated group is significant.compared to the control group with a *p-value. 0.05. Error bars represent ±SD.

Figure 14. Gel Concentrations vs. Stimuli Response (Trial 1). 15 earthworms in groups of 5 were treated in concentrations of clove oil gel ranging from 0%, 1%, to 2%. 2% of clove oil gel was the most effective in sedating the stimuli response of the earthworms at a fast rate as opposed to the 1% and 0% concentrations. Based on ANOVA following a Tukey HSD test, the 2% treated group is significant compared to the control group of 0% with a **p-value, 0.01. Error bars represent ±SD.

Figure 15. Immersion Stimuli Response vs. Gel Stimuli Response (Trial 1). 15 earthworms in groups of 5 were treated in concentrations of clove oil gel ranging from 0%, 1%, to 2%. There was no observable difference amongst the gel and immersion treated groups in sedating the earthworms at a fast rate for the 1% and 2% concentrations. Based on ANOVA following a Tukey HSD test, the 0% gel treated group is significant compared to 0% immersion with a *p-value, 0.05. ±SD.

Figure 16. Immersion concentrations vs. Mobility (normal movements) (Trial 1). 15 earthworms in groups of 5 were immersed in concentrations of clove oil treatment ranging from 0%, 1%, to 2%. The 1% and 2% were more effective at sedating the earthworms compared to the 0% group. Error bars represent ±SD.

Figure 17. Gel Concentrations vs. Mobility (normal movements) (Trial 1). 15 earthworms in groups of 5 were treated in concentrations of clove oil gel ranging from 0%, 1%, to 2%. The 2% concentration had the greatest number of movements over time with the gel application. Error bars represent ±SD.

Figure 18. Gel Concentrations vs. Convulsions (Trial 1). 15earthworms in groups of 5 were treated in concentrations of clove oil gel ranging from 0%, 1%, to 2%. Both the 1% and 2% concentrations had less convulsive movements than the 0% group. Error bars represent ±SD.

Figure 19. Immersion Mobility vs. Convulsions (Trial 1). 15 earthworms in groups of 5 were immersed in concentrations of clove oil treatment ranging from 0%, 1%, to 2%. Both the 1% and 2% concentrations had less convulsive movements than the 0% group. Error bars represent ±SD.

Figure 20. Immersion vs. Regeneration Length (Trial 1). 15 earthworms in groups of 5 were immersed in concentrations of clove oil treatment ranging from 0%, 1%, to 2%. All groups regenerated at a similar rate. The decrease in average length on Day 3 is due to the earthworms ejecting their tail. Error bars represent ±SD.

Figure 21. Gel vs. Regeneration Length. 15 earthworms in groups of 5 were treated in concentrations of clove oil gel ranging from 0%, 1%, to 2%. The length of the earthworms stayed consistent throughout the regeneration period of the worms. Error bars represent ±SD.

Figure 22. Immersion vs. Regeneration Mass. 15 earthworms in groups of 5 were immersed in concentrations of clove oil treatment ranging from 0%, 1%, to 2%. The length of the earthworms remains consistent throughout all concentrations. Error bars represent ±SD.

Figure 23. Gel vs. Regeneration Mass. 15 earthworms in groups of 5 were treated in concentrations of clove oil gel ranging from 0%, 1%, to 2%. All groups regenerated at a similar rate. There is a slight decrease in the average mass of the worms throughout the timespan. Error bars represent ±SD.

Trial 2

Figure 24. Immersion Concentrations vs. Response Time (Trial 2). 15 earthworms in groups of 5 were treated in concentrations of clove oil immersion ranging from 0%, 1%, to 2%. The response time of 1% concentration tended to be longer than both 2% and 0% immersion. Error bars represent ±SD.

Figure 25. Gel Concentrations vs Response Time (Trial 2). 15 earthworms in groups of 5 were treated in concentrations of clove oil gel ranging from 0%, 1%, to 2%. 2% of clove oil gel was the most effective in sedating the stimuli response of the earthworms at a fast rate as opposed to the 0% and 2% concentrations. Error bars represent ±SD.

Figure 26. Immersion Stimuli Response vs. Gel Stimuli Response (Trial 2). 30 earthworms in groups of 5 were treated in concentrations of clove oil gel or immersion ranging from 0%, 1%, to 2%. There was no observable difference amongst the gel and immersion treated groups in sedating the earthworms at a fast rate for the 1% and 2% concentrations. Based on ANOVA following a Tukey HSD test, the 0% gel treated group is significant compared to 0% immersion with a *p-value, 0.05. ±SD.

Figure 27. Mobility vs. Time (Immersion) (Trial 2). 15 earthworms in groups of 5 were immersed in concentrations of clove oil ranging from 0%, 1%, to 2%. There is no observable difference between the 0% and 1% concentrations in the sedation of the earthworms. There is an observable trend of a consistent decrease in the number of movements for all concentrations of clove oil. Error bars represent ±SD.

Figure 28. Mobility vs. Time (Gel) (Trial 2). 15 earthworms in groups of 5 were treated in concentrations of clove oil gel ranging from 0%, 1%, to 2%. There is no observable difference between the 0% and 1% concentrations in the sedation of the earthworms. There is an observable trend of a consistent decrease in the number of movements for all concentrations of clove oil. Error bars represent ±SD.

Figure 29. Number of Convulsive Movements vs. Time (Gel) (Trial 2). 15 earthworms in groups of 5 were treated in concentrations of clove oil gel ranging from 0%, 1%, to 2%. The 1% clove oil concentration had less convulsive movements than the 0% group throughout the period of recording. Moreover the 2% group showed no signs of convulsions throughout the 30 minute timespan. Error bars represent ±SD.

Trial 3

Figure 30. Immersion Concentrations vs. Response Time (Trial 3). 15 earthworms in groups of 5 were immersed in concentrations of clove oil ranging from 0%, 1%, to 2%. There is an observable trend that 1% immersion tended to have the most delayed response time. Error bars represent ±SD.

Figure 31. Gel Concentrations vs. Response Time (Trial 3). 15 earthworms in groups of 5 were treated in concentrations of clove oil gel ranging from 0%, 1%, to 2%. The 1% concentration was the most effective in sedating the response time of the earthworms at a fast rate as opposed to the 0% and 2% concentrations. Error bars represent ±SD.

Figure 32. Immersion Stimuli Response vs. Gel Stimuli Response (Trial 3). 30 earthworms in groups of 5 were treated in concentrations of clove oil gel or immersion ranging from 0%, 1%, to 2%. There was no observable difference amongst the gel and immersion treated groups in sedating the earthworms at a fast rate for the 1% and 2% concentrations. Nevertheless the immersion treated worms tended to respond at a slower rate as compared to the gel treated worms. Error bars represent ±SD.

Figure 33. Mobility vs. Time (Immersion) (Trial 3). 15 earthworms in groups of 5 were immersed in concentrations of clove oil ranging from 0%, 1%, to 2%. It can be observed that the 2% concentration of clove oil sedated the earthworms the most (SD error bars do not overlap). In other words the amount of limited movements was limited for the 2% group of earthworms. Error bars represent ±SD.