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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 6  |  Issue : 2  |  Page : 80-84

Basil (Ocimum basilicum) Leaves Essential Oil Ameliorates GluR1 Receptor Expression, TNF-α Level, and Pain-like Behaviors in Post-operative Pain Setting


1 Department of Anesthesiology and Intensive Care, Faculty of Medicine, Bali, Indonesia
2 Department of Pharmacology, Faculty of Medicine, Bali, Indonesia
3 Department of Virology and Immunology, Faculty of Veterinary Medicine, Udayana University, Bali, Indonesia

Date of Submission02-Feb-2022
Date of Decision04-Mar-2022
Date of Acceptance11-Mar-2022
Date of Web Publication09-May-2022

Correspondence Address:
Dewa Ayu Mas Shintya Dewi
Department of Anesthesiology and Intensive Care, Faculty of Medicine, Udayana University, Jl. PB Sudirman, Denpasar 80232, Bali
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bjoa.bjoa_39_22

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  Abstract 

Background: Unrelieved post-operative pain is an emerging healthcare concern with ever increasing global volume of surgical procedures. GluR1 subunit coupled with tumor necrosis factor (TNF)-α expression plays a major role in the development of post-operative pain mediated by α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor. There was no existing evidence on the analgesic potential of basil essential oil (BEO) in post-operative settings, despite its well-established antinociceptive and anti-inflammatory activities. Materials and Methods: BEO was subjected to gas chromatography–mass spectrometry (GC–MS) analysis to identify the active ingredients. The antinociceptive and anti-inflammatory activities of orally administrated basil (Ocimum basilicum) essential oil were tested in a rat model of post-operative pain using hindpaw surgical incision as noxious stimuli. TNF-α and GluR1 subunit expressions were measured using enzyme-linked immunosorbent assay and immunohistochemistry methods. Spontaneous pain and mechanical hyperalgesia were measured using mouse grimace scale and Von Frey monofilament test, respectively. All outcomes were evaluated in acute post-operative pain timeframe. Results: Chemical analysis identified 14 terpenoids predominated with caryophyllene and citral. BEO administration caused a significant reduction of TNF-α (67.23 ± 2.46 vs. 70.45 ± 4.89; P = 0.019) and GluR1 (3.03 ± 0.56 vs. 3.90 ± 1.12; P = 0.005) levels at 24 h after surgical incision when compared with the control group. Significant spontaneous pain, pain threshold, and pain-like behaviors frequency reduction at 1-, 4-, and 24-h post-surgical incision were also noted. Conclusion: Effective antinociceptive activity of BEO through modulation of GluR1 and TNF-α levels was further confirmed in the behavioral outcome. Advancement into clinical translation necessitates BEO pharmacological profiling, especially given the diversity of chemotypes.

Keywords: AMPA, enzyme-linked immunosorbent assay, hyperalgesia, pain, post-operative, receptors


How to cite this article:
Dewi DA, Jawi M, Astawa NM, Ryalino C. Basil (Ocimum basilicum) Leaves Essential Oil Ameliorates GluR1 Receptor Expression, TNF-α Level, and Pain-like Behaviors in Post-operative Pain Setting. Bali J Anaesthesiol 2022;6:80-4

How to cite this URL:
Dewi DA, Jawi M, Astawa NM, Ryalino C. Basil (Ocimum basilicum) Leaves Essential Oil Ameliorates GluR1 Receptor Expression, TNF-α Level, and Pain-like Behaviors in Post-operative Pain Setting. Bali J Anaesthesiol [serial online] 2022 [cited 2022 May 26];6:80-4. Available from: https://www.bjoaonline.com/text.asp?2022/6/2/80/344883




  Introduction Top


Post-operative pain management should be considered a strategic priority for healthcare service provision, whereas global volume of surgical procedures handled yearly surge from 234 to 313 million within a decade up to 2015.[1],[2] Recent review indicated that at least 80% of patients who undertook surgery suffered from post-operative pain of variable intensity and over three-quarters of those suffered from pain of moderate or worse intensity.[3] Latest analysis of 50,005 patients from 26 countries in the PAIN-OUT post-operative acute pain registry confirmed the figure, with patient-reported median worst pain intensity of 5–6 in the studied cohorts.[4],[5],[6]

Current basic scientific evidence pointed to the role of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor in spinal sensitization after surgical incision as the main non-N-methyl-d-aspartate receptor group alternative.[6] The AMPA receptor as a member of ionotropic glutamate receptors functional classes is further classified into GluR1 to GluR4, where the former two play a major role in the fast excitatory transmission. Although subunit combination along with glutamate release and uptake timing contributes to the kinetics and amplitude of excitatory synaptic response, the most reasonably modifiable and measurable factor in the equation is the density of receptor expression and thus serves as a surrogate outcome for the excitotoxicity potential.[7],[8],[9]

Multimodal approaches to post-operative pain management are the cornerstone of state-of-the-art treatment strategy.[10],[11] These approaches embrace full breadth of pharmacological and non-pharmacological interventions with the main goal of effectively managing acute pain and preventing further sensitization.[10],[12] Natural essential oils have the propensity to be pharmacological adjuvant given their intrinsic anti-inflammatory and antinociceptive activities.[13] Basil (Ocimum basilicum)-derived products have been reported to demonstrate both activities; however, the scientific rigor employing surgical incision technique and evaluating pain-like behaviors is lacking.[14],[15],[16] The present study was designed to investigate the effect of basil essential oil (BEO) on GluR1 receptor expression, serum tumor necrosis factor (TNF)-α level, and pain-like behaviors in acute post-operative pain settings using rat experimental model simulation of surgical incision.


  Materials and Methods Top


Male 8- to 12-week-old Wistar rats (Rattus norvegicus) weighing 180–200 g were kept in the local animal containment facility within the university laboratory. They were housed individually as appropriate with ad libitum access to food and water and maintained in a 12-h light/dark cycle (lights on at 08:00 and lights off at 20:00). The number of animals used was limited to the minimum number required to obtain significancy, and randomly assigned treatment was done only once per animal. The experiment procedures were designed and conducted to minimize suffering to animals and fully complied to the protocol approved by the Institutional Review Board (letter no. B/71/UN14.2.9/PT.01.04/2021, dated April 1, 2021).

Essential oil preparation and analysis

The leaves of O. basilicum were identified by the Indonesian Institute of Sciences and then dried for at least 4 consecutive days until no weight loss was observed before proceeding to pure steam distillation process. Essential oil was isolated by forcing hot steam (100–110°C) through the samples, thus volatilizing and extracting the oil, which will then be cooled through the condenser together with the water to enter the liquid phase. Resulting products consisting of volatile oil as the upper phase and hydrosol as the lower phase were separated based on their immiscibility. Gas chromatography coupled with mass spectrometry (GC–MS) analysis was performed on a GC-MS HP-5 column (0.25 μm d.f. × 0.25 mm i.d., 20 m) model GCMS-QP2010 Ultra (Shimadzu Corporation, Kyoto, Japan).

Experimental plan

The animals were allocated randomly with 1:1 ratio into two experimental groups, namely, treatment and control groups. Animals in the treatment group were treated with orogastric gavage feeding of 400 mg/kg BEO, whereas those in the control group were treated with 2 mL sterile 0.9% sodium chloride solution. All treatments were commenced an hour prior to surgical incision. The animals underwent general anesthesia with 2% isoflurane, and the plantar incision was done as described in the original model.[17]

Evaluation of serum TNF-α level

Serum TNF-α level was measured by the rat enzyme-linked immunosorbent assay (ELISA) kit closely following manufacturer’s instruction (Fine Test, Wuhan, China). Briefly, TNF-α containing serum was collected from orbital sinus blood sampling at 24 h after surgical incision and poured onto the plate to combine with biotinylated ELISA assay and streptavidin conjugated with horseradish peroxidase. Automated plate reading by spectrophotometric detection of tetramethylbenzidine at 450 nm was performed.

Evaluation of GluR1 expression

The animals were euthanized using isoflurane promptly after blood sampling, and hydraulic extrusion of the spinal cord was performed. The spinal cord preparation underwent rinsing, dehydration, and paraffin embedding followed by sectioning into µm thick blocks. The blocks were subjected to immunohistochemical staining using primary and secondary antibodies (Histofine Simple Stain MAX-PO; Nichirei Bioscience, Tokyo, Japan) ensued with diaminobenzidine tetrahydrochloride chromogen staining. Visualization of blocks corresponding to L1-L2 levels was done under a light microscope at 400× magnification.

Post-operative pain

Baseline and post-operative pain assessments of both spontaneous pain and mechanical hyperalgesia were captured before and at 1, 4, and 24 h after the surgical procedure. The facial expressions captured using a Sony Alpha 7 II camera were saved in JPEG format and codes were assigned for blinding purpose. Spontaneous pain was gaged by assessing the relative difference in post-operative mouse grimace scale (MGS) score from the baseline. Mechanical hyperalgesia was tested using the percent response method of the manual Von Frey test. The monofilaments in the ascending order of thickness (0.04-, 0.40-, 1.4-, 2.0-, 4.0-, 6.0-, 8.0-, 10.0-, 15.0-, and 26.0-g bending force) were used to apply perpendicular pressure with predetermined force in the vicinity of incision. Each filament thickness was applied five times per second with 2–3-s interval regardless of the response. Positive response represents pain-like behaviors comprising paw withdrawal, licking, or shaking. Mechanical threshold was determined when 50% or above positive responses of any identical nocifensive behaviors were elicited, whereas nocifensive behavior frequency was expressed as a percentage of positive responses for each force.

Statistical analysis

Data presentation was designated as mean ± SD or median (interquartile range) as appropriate. Data were analyzed using the t-test and Mann–Whitney as indicated. Differences were considered statistically significant when P < 0.05.


  Results Top


Fourteen terpenoids were characterized in the BEO by GC–MS analysis and were listed in [Table 1] in the order of their abundance. Majority of terpenoids constituted of sesquiterpenoids (39.49%) as opposed to monoterpenoids (28.52%). Caryophyllene and citral were the most abundant sesquiterpenoid and monoterpenoid, respectively. Caryophyllene, citral, linalool, germacrene, and aromandrene were considered to be the main compounds contributing to nearly half of the total constituents. Non-terpenoid compounds accounted for 31.99% of the constituents.
Table 1: Terpenoids identified in the basil essential oil

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Assessment of TNF-α and GluR1 expression at 24 h yielded significantly lower result in the treatment group [Table 2]. Visualization of GluR1 expression is performed using indirect immunohistochemistry and diaminobenzidine tetrahydrochloride chromogen staining. [Table 3] shows the between-group comparison of spontaneous pain and mechanical hyperalgesia in baseline pre-operative and at 1-, 4-, and 24-h post-operative settings. Baseline pain assessment results for all parameters were not statistically different. Spontaneous or non-stimulus-evoked pain as measured by MGS and mechanical stimulus-evoked pain threshold and pain-like behavior frequency were significantly lower in the treatment group at all time points.
Table 2: Effects of BEO administration on serum TNF-α level and GluR1 expression

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Table 3: Comparison of acute post-operative pain assessments

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  Discussion Top


This study was the first to investigate the analgesic effect of BEO on combined laboratory and behavioral parameters, particularly in the post-operative setting. There were only two other pre-clinical studies using BEO identified in the latest systematic review and meta-analysis.[18] Additionally, the result of BEO compound analysis could fill the gap in the knowledge of O. basilicum heterogenous chemotypes.

The qualitative and quantitative variability of BEO chemical composition was notable from earlier studies. Interestingly, the major terpenoids found in the current chemical analysis (i.e., caryophyllene and citral) did not match the four preexisting chemotypes, each of which was identified by high amounts of linalool, estragole, methyl cinnamate, or eugenol.[14],[19] However, similar constituents, despite different relative amounts, were found in a study with geographical proximity.[20] A systematic review suggested the immunomodulatory and local anesthetic-like activities of caryophyllene aside from its neuroprotective potential.[21] The anti-inflammatory, immunomodulatory, antihyperalgesic effects of citral were also discovered more recently, further highlighting the synergistic potential of both terpenoids in post-operative pain management.[22],[23]

Induction of pain following peripheral noxious stimuli is closely regulated by the specific subunit phosphorylation of GluR1 and its interaction with partner proteins in the dorsal horn neurons.[24] Accumulated evidence from prior studies showed that the mechanism by which TNF-α was involved in GluR1 subunit upregulation at the post-transcriptional level, especially through TNF-α receptor 1 (TNFR1), resulted in net increase in (1) extracellular glutamate levels by inducing its exocytosis and inhibiting its uptake and (2) excitatory synaptic strength by preferentially acting on Ca2+ permeable-AMPA receptors.[8] These co-existing roles warrant both GluR1 and TNF-α to be investigated and mitigated simultaneously.

Analgesic effect of inhalation and intraperitoneal injection of BEO were reported previously at different doses for up to 180 mg/kg using multiple non-surgical noxious stimuli. The study also confirmed the involvement of delta- and mu-opioid pathways upon exerting its effect.[25] Since both delta- and mu-pathways were known to be located upstream of the GluR1 trafficking cascade, it served as one plausible mechanistic explanation supporting current findings of decreased GluR1 and TNF-α levels.[26],[27]

Although both previous studies identified in the latest systematic review and meta-analysis used up to 200 mg/kg dose of BEO, we justified our 400 mg/kg in the light of another study, which tested up to 160 mg/200 g dose (equivalent to 800 mg/kg) and reported similar anti-inflammatory activity at 80 mg/200 g dose (equivalent to 400 mg/kg).[28] Route of administration should be put into consideration as the current study was the only one opting for oral route. Additionally, the predetermined dose in this study was well below the lethal dose of 50% death (LD50) of 532 mg/kg.[16]

The chemical analysis in our BEO formulation enriched the preexisting body of evidence on chemotype variation. Intrinsically variable composition of BEO per se should elicit thorough investigation on which particular chemotype or active ingredient yields more desirable results and gives rationale for future plant preparation standardization. Although the study design was not intended to explore the pharmacodynamics of BEO, the interconnection of current study findings with previously identified pathways unraveled an avenue for future pharmacodynamics study. Lastly, pharmacological profiling is required to allow for translation into robust clinical evidence and praxis.


  Conclusion Top


The BEO oral administration in this pre-clinical study effectively counteracted surgical incision-induced nociception through the modulation of GluR1 and TNF-α levels. Significantly reduced pain-like behavior further confirmed the laboratory investigation findings. Future studies on BEO pharmacological profile are prerequisite for advancement into clinical translation.

Authors’ contributions

The authors contributed equally in all study process and manuscript preparation, editing, and review.

Financial support and sponsorship

None to declare.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2], [Table 3]



 

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