Introduction

Anxiety related to dental treatment can generate physiological, cognitive and behavioral effects. In particular, impacted third molar extraction is often associated with peaks of anxiety, as it is an invasive and complex procedure that requires time for recovery. As such, it generates a negative expectation, which is aggravated by the fact that the patient is losing a tooth Sharif MO1.

An alternative to overcome the effects of this anxiety lies in benzodiazepines, as these can generate conscious sedation and possess an anxiolytic effect through depression in the central nervous system. With correct use, there is a low level of complications Jerjes W, Jerjes WK, Swinson B et a.l2. The most commonly used substance is midazolam, but despite its low toxicity patients may experience adverse reactions like skin rash, nausea or headache. Against this background, there has been an increase in the search for alternatives with lower risks and side effects, but still capable of maintaining stress control; herbal medicines for instance Miller CS, Dembo JB, Falace DA et al.3.

In many cases, the clinical impression alone can alert the dentist to a patient’s fear or anxiety, which may be confirmed through subjective analyses like formalized questionnaires Freeman RE.4. One example is the Modified Dental Anxiety Scale (MDAS) questionnaire for adults, a questionnaire with only five items that assesses a patient’s dental anxiety levels Corah NL.5. Another widely used tool is the Corah Dental Anxiety Scale (CORAH) scale of 1969, which subjectively assesses the patient’s anxiety levels Corah NL.5. Other scales, such as the Hospital Anxiety and Depression Scale (HAD), developed by Zigmond AS, Snaith RP.6, are for hospital use and take into account the patient’s emotional and personal aspects.

Despite their convenience and accessibility, these surveys and scales have some crucial shortcomings that limit knowledge about the patient’s psychological state Le SH, Tonami K, Umemori S et al.7. First, even though each dental procedure has its own characteristics, these anxiety scales have been developed for general examinations and do not focus on any specific treatment Astramskaitė I, Poškevičius L, Juodžbalys G.; Carter AE.8,9. Second, the fear of dental treatment is most commonly based on a combination of personal characteristics, the patient’s current anxiety state, social status and coping ability, rather than a specific “dental anxiety”7,9.

Therefore, to assess patient stress and anxiety levels and reduce research bias, objective methods such as biochemical and physiological analyses can be employed Le SH, Tonami K, Umemori S et al.7. Aiming at a quantitative analysis of anxiety, we can use circulatory and respiratory markers like heart rate (HR), blood pressure (BP) and blood oxygen saturation (SpO2), all of which are excellent non-invasive and relatively easy to use methods2.

Nonetheless, the assessment of basic physiological parameters in itself can generate anxiety in patients. Biochemical analyses of several factors, such as adrenocorticoid hormones, serotonin and cortisol in serum are recommended, but the mere sight of blood and the act of taking samples from the body can induce stress and influence the readings Agarwal N, Chaturvedy S, Chaturvedi S10. A possible means to overcome this obstacle is the use of saliva, a potential alternative and universal diagnostic fluid that can be collected non-invasively with little training and without side effects Aardal & Holm11. An increase in serum cortisol production leads to a proportional increase in its levels in saliva. Consequently, salivary concentrations are closely correlated Aardal E Holm AC.; Sapolsky RM, Krey LC, Mcewen BS.11,12. Cortisol is a glucocorticoid hormone secreted by the adrenal cortex, which regulates the metabolism of lipids, water, proteins and carbohydrates, maintains vascular reactivity, affects the sensitivity of the nervous system, regulates the number of blood cells and affects human stress levels3,13.

Phytotherapy has long been used to treat depressive illnesses, especially in patients for whom the use of benzodiazepines is contraindicated, whether due to drug resistance, drug interference or allergy. It is now being studied as an alternative treatment for anxiety disorders Kinrys G, Coleman E, Rothstein E.14. Passiflora incarnata, the “Passionflower” is a plant found in tropical regions, commonly used in traditional medicine to treat anxiety, nervous breakdowns and neuralgia Dhawan K, Kumar R, Kumar S, Sharma A; Ernst E.15,16. In addition to its lower cost, phytotherapy has a lower risk of side effects and dependence13.

Although Passiflora incarnata has been used as a sedative and anxiolytic in dental practice de Moares MB, Barbier WS, Raldi FV t al.; Kaviani N, Tavakoli M, Tabanmehr M et al. 13,17. Moreover, the literature still lacks robust evidence regarding the application of anxiety assessment methods in the context of impacted third molar surgery. The present study thus underscores the importance of investigating effective alternatives for anxiety management in this specific scenario, particularly for patients with contraindications to benzodiazepine use.

Objectives

The objective of this triple-blind, parallel, paired and randomized clinical study was to evaluate the performance of Passiflora incarnata (PAS) in controlling dental anxiety in impacted impacted third molar extraction surgeries, using a negative (placebo-PLA) and positive control (midazolam-MID).

Methodology

Experimental design and ethical details

This study consists of a triple-blind, parallel, randomized clinical trial that began following approval by the Research Ethics Committee of the Dentistry School of Araçatuba (case number 053966/2021- Certificate of Presentation for Ethical Assessment 47,159,621.0.0000.5420). All participants signed a Free and Informed Consent Form Butcher NJ, Monsour A, Mew EJ et al.18. The study was also submitted to the Brazilian Clinical Trials Registry platform—ReBEC (RBR-4knsh9z) and conducted in accordance with the CONSORT guidelines (www.consort-statement.org) and the 1963 Helsinki Declaration19.

Subjects

The study initiated with an interview and evaluation of 75 patients. Of these, 30 did not meet the inclusion criteria. Of the remaining 45, five presented complications during or after the surgery, such as bleeding, infections and failure in tooth extraction, totaling 40 operated patients. Given that another 10 did not return for the postoperative follow-up, in total 30 patients were included in the study (Fig. 1A).

Fig. 1
Fig. 1
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A- Flowchart of the individuals selected according to CONSORT guidelines. Initially, 75 patients were screened; of these, 30 did not meet the inclusion criteria, resulting in 45 eligible patients. Among them, 5 experienced postoperative complications and were excluded, totaling 40 patients who underwent the surgical procedure. Of these, 10 did not attend the postoperative follow-up, thus 30 patients were included in the final analysis. B-Illustrative sequence of the standardized surgical procedure performed for the removal of impacted mandibular third molars. The images represent: (A) panoramic radiograph showing a patient eligible for the study; (B) initial clinical view of the surgical site; (C) exposure of the anatomical structure of interest; (D) performance of the osteotomy; and (E) tooth sectioning (odontosection). All procedures were carried out by a trained surgical team, following a standardized protocol to ensure consistency and reproducibility across cases.

Thus, 30 healthy young adults of both sexes, aged 16 to 35, were selected as participant and treated at the surgery clinic of the Dentistry School of Araçatuba.

The inclusion criteria were determined through anamnesis, clinical and radiographi) Jerjes W, Jerjes WK, Swinson B et al.; de Moares MB, Barbier WS, Raldi FV et al.; Antunes AA, Avelar RL, Martins Neto EC et al.;Steed MB.2,13,20,21: Patients in good systemic and local health conditions, aged 16 to 35 with indication for the extraction of Impacted third molar (elements 18, 28, 38 or 48) and at least 2/3 of the root formed, according to the radiographic evaluation, thus belonging to classification I or II and position A or B, as per Pell and Gregory22.

The following exclusion criteria were also verified through anamnesis, clinical and radiographic examinations Jerjes W, Jerjes WK, Swinson B et al.; Miller CS, Dembo JB, Falace DA et al. ; Le SH, Tonami K, Umemori S, et al.;Antunes AA, Avelar RL, Martins Neto EC et al Steed MB; Pereira-Santos D, Brêda-Júnior MA, Ferraz EP et al.2,3,7,20,21,22,23; presence of third mandibular molars in positions 3 and C according to the Pell and Gregory classification Santosh P22; a presence of any local manifestations in the area of interest that may contraindicate the surgical procedure, such as pericoronitis, odontogenic cysts and tumors associated or not with the impacted impacted third molar, trauma in the region, or any symptoms that indicate the presence of infection; patients who smoke or have some type of systemic disease (of any organic system) like diabetes, systemic arterial hypertension, hyperthyroidism, osteoporosis, gastrointestinal diseases that compromise the outcome of the surgery or that contraindicate the administration of drugs used in the research; patients with a history of hypersensitivity to any drug used in the research; intolerance to other materials used in the research, such as 0.5% chlorhexidine alcohol solution, 0.12% chlorhexidine digluconate solution and 2% mepivacaine hydrochloride solution with 1:100,000 epinephrine; female patients in their menstrual period, pregnant or lactating during the surgical procedures; patients with an abnormal sleep pattern who slept less than 5 h the night before surgery; individuals on medication to control psychiatric illnesses; and, finally, those who were taking medications that interfere with cortisol analyses, such as corticosteroids, estrogens and androgens.

Sample size

To define the number of patients required for the study, the sample size was calculated using the “Sample Size for ANOVA” tool of the SigmaPlot 12.0 statistical program. For this purpose, the results of a primary outcome analysis (diastolic blood pressure) were used, with statistical difference between the groups, using as reference the work developed by de Moares et al. (2019). The calculation included a difference between means of 0.95 and a standard deviation of 1.03, with a significance level of 95% and α = 0.05, thus obtaining n = 9 individuals per group. The sample size was increased to n = 10 per group, due to the risk of having to exclude a patient at a later stage or a patient failing to return during the course of the research, in accordance with the literature24,25,26,27.

Tested groups, randomization, surgical protocol, and blinding method

After inclusion, the patients were divided into the following 3 groups (n = 10) according to the preoperative medication that was administered, all orally Jerjes W, Jerjes WK, Swinson B et al.; Dantas L, de Oliveira-Ribeiro A, de Almeida-Souza L et al.; Garip H, Gürkan Y, Toker K et al. 2,28,29: 1-MID (7.5 mg of midazolam); 2-PLA (Placebo); and 3-PAS (260 mg of Passiflora incarnata- Prepared product in a specialized compounding pharmacy in the municipality of Araçatuba, São Paulo.).

Midazolam is available on the market in doses of 7.5 and 15 mg. The choice for the 7.5 mg dose was made based on the literature, which has demonstrated that this dose has a significant anxiolytic effect and is far below the maximum dose of 20 mg13,30.

The medications were administered 30 min prior to surgery, so as not to affect the application of the questionnaires. All participants underwent the surgical procedure by the same surgeon and assistant, who were blinded to the medication provided to the patients. In all surgical interventions, vestibular osteotomy and the necessary odontosection for tooth removal were performed.

A fourth researcher was responsible for randomizing the pharmacological group to which the patient was submitted. He randomly selected the patients using the envelope system, which had 3 papers identifying the groups 1—MID, 2—PLA and 3 – PAS. In this way, the groups were assigned while the same researcher was responsible for providing the medication protocol to the patients.

When a patient had bilateral impacted third molars, the choice of the right or left side to be initially operated on was also determined by the same researcher, using the envelope system. There were two papers identifying the right (18 and 48) and left side (28 and 38). The surgeon, the patient and the researcher who performed the analyses were all blinded to the administered drug in order to ensure the reliability of the study as triple blind (Fig. 2).

Fig. 2
Fig. 2
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Schematic representation of the main stages of the experimental protocol adopted in the study. The timeline illustrates the sequence of procedures performed: initial measurement of vital signs; administration of evaluation questionnaires; baseline saliva collection; 30-min interval before medication administration; medication administration; beginning of the surgical procedure; intraoperative measurements (heart rate, blood pressure, and pulse oximetry) taken at the following time points: preoperative, beginning of surgery, anesthesia, incision, extraction, suturing, and postoperative; immediate postoperative saliva collection; new measurement of vital signs; and re-administration of questionnaires 30 min after the end of surgery. All procedures were conducted according to a previously standardized protocol, ensuring consistency and comparability among participants.

Surgical phase

The surgical procedures were performed by the same operator. When a patient had bilateral Impacted third molar, the surgeries were performed unilaterally with a minimum interval of 21 days between one surgery and the other28,31,32.

All patients underwent intraoral antisepsis with an aqueous solution of 0.12% chlorhexidine digluconate, thoroughly rinsing the mouth with the solution for 1 min, followed by extraoral antisepsis with an aqueous solution of 0.5% chlorhexidine.

The employed anesthetic technique was a regional blockade of the inferior alveolar nerve (buccal and lingual) by applying 2% Mepivacaine Hydrochloride with epinephrine 1:100,000, with a maximum volume of up to 4.5 ml, equivalent to that contained in two and a half tubes.

A linear triangular mucoperiosteal incision was made with a number 15 scalpel blade in the distal region of the 2nd lower molar, associated with a vestibular relaxing incision in the mesial region of the 2nd molar. Subsequently, mucoperiosteal detachment was performed. The osteotomy was performed using a truncated conical carbide bur n. 702 mounted on a handpiece. Tooth removal was completed with curved and straight extractors, followed by a careful inspection to remove the pericoronal follicle. The bone edges were filed to remove the bone spicules with a bone file, whereafter abundant irrigation was performed with sterile 0.9% NaCl saline solution. After this surgical step, interrupted sutures were put in place using 5.0 nylon thread. The surgeries were performed in the morning (between 8 and 10 am), so that there would be no interference from diurnal variations in cortisol levels2,3 (Fig. 1B).

Postoperative phase

Thirty minutes post-surgery, measurements of the physiological parameters, saliva collection and application of questionnaires were repeated. The patients were instructed to stick to a liquid, soft, high-protein and cold diet for the first 48 h after surgery, in addition to other general precautions, such as avoiding exposure to the sun or heat, avoiding physical strain and not using any type of mouthwash for the first 24 h. They were also advised to undergo cryotherapy for the 24 to 48 h after surgery.

The participants received twelve 750 mg paracetamol tablets and were instructed to take 1 tablet only in case of pain, respecting a minimum interval of 6 h. All patients received prophylactic antibiotic therapy with amoxicillin 500 mg every 8 h for 7 days or, in case of allergy, clindamycin 300 mg every 8 h for 7 days. The patients used another analgesic (Dipyrone 1 g) only in cases of allergy to acetaminophen.

Variables

The predictor variable was the level of anxiety assessed before and after impacted third molar surgeries.

Primary outcomes

The physiological parameters of the patients (HR, BP and SpO2), as well as salivary biochemicals constituted the primary outcomes of this study.

The physiological parameters were measured pre-, intra- and postoperatively. Measurements were taken 30 min before the start of the procedure, and during the procedure at the following stages: start of the procedure; anesthesia; incision; extraction of the element; and suture Jerjes W, Jerjes WK, Swinson B et al.;Dantas L, de Oliveira-Ribeiro A, de Almeida-Souza L et al.2,28. Postoperative measurement took place 30 min after the end of the operation. BP was measured using a sphygmomanometer and stethoscope. HR and SpO2 were measured using a digital oximeter.

Saliva was collected according to a protocol adapted from dos Santos DR, Souza RO, Dias LB et al.33. Unstimulated whole saliva samples were collected from all participants. They were instructed to brush their teeth and not to ingest any food or drink two hours prior to saliva collection. Pre-surgery, they rinsed their mouths with water, after which unstimulated whole saliva was expectorated every 60 s and collected in sterile plastic tubes at a volume of 5–8 ml. Saliva secreted in the first 60 s was discarded.

Postoperative collection was performed 30 min after surgery to avoid contamination with blood. Any saliva with traces of blood was discarded. The samples were stored in boxes with ice and transported to the laboratory within one hour for storage. The samples were then centrifuged for 10 min at 3000 rpm and a temperature of 4 °C, after which the supernatants were stored at −80 °C. Biochemical analyses were performed up to 15 days after collection.

Salivary cortisol levels were measured in all patients through chemiluminescence assay according to the manufacturer’s specifications (Elecsys and Cobas Analyzers®, Roche, Roche Austria GmbH Engelhorngasse 3 1211 Vienna,Austria). The results were expressed in µg/dl.

Furthermore, the enzymatic activity of α-amylase was measured in all patients, using the method described by Caraway WT; Howe L, Elmslie RG  34,35. A mixture of soluble starch 0.04% (m/v) in phosphate buffer (pH 7.0) and saliva was incubated at 37 °C for 7.5 min so that the α-amylase present in the samples could promote the hydrolysis of the polysaccharide. After incubation, an aliquot of the solution containing potassium iodate and hydrochloric acid was added to this mixture to stop the reaction. The absorbances were determined at a wavelength of 660 nm, and the results expressed as U/L.

Additionally, the concentration of total salivary proteins in the saliva aliquots was determined by the bicinchoninic acid (BCA) method, in accordance with the manufacturer’s recommendations. The solution was homogenized and read at an absorbance of 660 nm using a spectrophotometer. The results were expressed in g/L.

Secondary outcomes

Questionnaires to assess the level of dental anxiety Freeman RE; Corah NL; Zigmond AS, Snaith RP.4,5,6 were determined as secondary outcomes. The Corah scale, the HAD hospital scale and the MDAS were the parameters used in this study. This combination of three questionnaires was employed to broaden the view on subjectivity of the anxiety assessment. They were given to the patients 30 min before surgery in order to avoid medication influence, and 30 min after the end of the procedure2,13,28.

It is worth noting that the MDAS and Corah instruments do not have copyright restrictions for use. However, the use of the HAD questionnaire requires prior authorization. For this study, the Master User License Agreement was requested from the MAPI Research Trust, which granted permission for non-commercial use and without modifications.

Statistical analysis

For statistical analysis, the present study used SigmaPlot 12.0 (Systat Software, Inc, v.12, Chicago, IL, USA). The Shapiro–Wilk normality test was applied to all parameters (P > 0.05). Absolute values were submitted to the two-way ANOVA test (group vs. time), and the Holm-Sidak post-test in case the P-value was lower than 0.05. For all tests, the significance level was set at 5% and 95% confidence intervals.

Results

One patient suffered complications during the experiment. A postoperative hemorrhage occurred, which was immediately treated, and the patient was excluded from the study and replaced by another individual.

Physiological data

Heart rate

Heart rate (HR) was monitored throughout the perioperative period to assess the physiological response to anxiety. No statistically significant differences were observed between groups at any time point (P > 0.05). During the preoperative period, mean HR values were 87.6 ± 12.19 (MID), 83.0 ± 18.91 (PLA), and 78.3 ± 10.86 (PAS). At the start of surgery, values were 80.33 ± 9.58 (MID), 74.86 ± 18.01 (PLA), and 77.75 ± 7.96 (PAS). During anesthesia, measurements were 92.4 ± 14.53 (MID), 85.4 ± 20.71 (PLA), and 89.31 ± 16.76 (PAS). The incision phase showed values of 89.46 ± 12.87 (MID), 79.33 ± 61.90 (PLA), and 85.06 ± 13.47 (PAS). Extraction phase values were 74.86 ± 11.62 (MID), 74.86 ± 15.43 (PLA), and 81.56 ± 12.89 (PAS). During suture, measurements were 80.26 ± 8.49 (MID), 75.0 ± 14.76 (PLA), and 78.37 ± 10.77 (PAS). Postoperative values were 78.06 ± 10.06 (MID), 73.13 ± 9.86 (PLA), and 80.75 ± 11.75 (PAS). The consistent lack of significant differences suggests that none of the interventions elicited differential effects on this autonomic parameter under the studied conditions (Fig. 3 A).

Fig. 3
Fig. 3
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Graphical representation of the results obtained from the measurement of physiological parameters during the surgical procedure. The parameters evaluated were: (A) heart rate, (B) systolic blood pressure, (C) diastolic blood pressure, and (D) peripheral oxygen saturation (SpO₂). Each panel shows the values recorded at the following experimental time points: preoperative, beginning of surgery, anesthesia, incision, extraction, suturing, and postoperative. No statistically significant differences were observed between groups for heart rate, systolic blood pressure, or peripheral oxygen saturation (p > 0.05) at any of the time points. However, a statistically significant difference was found between the MID and PLA groups for diastolic blood pressure at the time of anesthesia (p < 0.05).

Systolic blood pressure

Systolic blood pressure (SBP) analysis revealed similar values among groups across all perioperative stages (P > 0.05). Pre-operative values were 118.0 ± 6.76 (MID), 116.6 ± 15.8 (PLA), and 117.5 ± 12.9 (PAS). At the start of surgery, measurements were 115.3 ± 11.8 (MID), 120.6 ± 10.9 (PLA), and 116.2 ± 12.5 (PAS). During anesthesia, values were 118.0 ± 10.1 (MID), 122.0 ± 13.2 (PLA), and 120.6 ± 16.1 (PAS). The incision phase recorded 115.3 ± 11.8 (MID), 118.6 ± 12.4 (PLA), and 118.75 ± 15.0 (PAS). Extraction stage values were 112.6 ± 10.9 (MID), 122.0 ± 14.2 (PLA), and 116.8 ± 15.7 (PAS). During suture, measurements were 114.0 ± 12.44 (MID), 118.6 ± 11.8 (PLA), and 114.3 ± 15.9 (PAS). Post-operative values were 108.6 ± 10.6 (MID), 118.0 ± 11.4 (PLA), and 112.5 ± 16.9 (PAS). The absence of significant intergroup variations suggests comparable effects of all interventions on systolic blood pressure regulation during impacted third molar surgery (Fig. 3B).

Diastolic blood pressure

Diastolic blood pressure (DBP) analysis revealed no statistically significant intergroup differences among the MID, PLA, and PAS groups across most perioperative stages (P > 0.05). Pre-operative values were 71.3 ± 6.3 (MID), 73.8 ± 6.2 (PLA), and 75.6 ± 8.9 (PAS). At the start of surgery, measurements were 69.3 ± 7.9 (MID), 71.3 ± 6.3 (PLA), and 74.3 ± 7.2 (PAS). However, during anesthesia, PAS (73.1 ± 7.04) was higher than MID (66.0 ± 7.3) (P < 0.05), though no significance was found between PLA (70.0 ± 7.5) and MID or between PLA and PAS. At incision, values were 65.3 ± 7.4 (MID), 68.6 ± 7.4 (PLA), and 70.6 ± 8.5 (PAS). Extraction stage measurements were 66.6 ± 6.1 (MID), 71.3 ± 9.1 (PLA), and 71.8 ± 8.3 (PAS). During suture, values were 70.0 ± 6.3 (MID), 70.0 ± 9.2 (PLA), and 71.8 ± 8.3 (PAS). Post-operative measurements were 74.6 ± 7.7 (MID), 74.6 ± 9.9 (PLA), and 73.7 ± 8.8 (PAS). The isolated significant difference during anesthesia suggests a transient differential effect between Passiflora incarnata and Midazolam on diastolic blood pressure, though this effect was not sustained in subsequent phases (Fig. 3C).

Blood oxygen saturation

Blood oxygen saturation (SpO₂) remained stable and within physiological limits across all groups throughout the surgical procedure, at any time point (P > 0.05). Pre-operative values were 98.0 ± 0.01% (MID), 97.0 ± 0.01% (PLA), and 97.0 ± 0.01% (PAS). At the start of surgery, measurements were 98.0 ± 0.009% (MID), 97.0 ± 0.01% (PLA), and 98.0 ± 0.01% (PAS). During anesthesia, values were 98.0 ± 0.009% (MID), 98.0 ± 0.008% (PLA), and 98.0 ± 0.008% (PAS). The incision phase recorded 98.0 ± 0.008% (MID), 98.0 ± 0.01% (PLA), and 97.0 ± 0.02% (PAS). Extraction stage values were 98.0 ± 0.005% (MID), 97.0 ± 0.01% (PLA), and 97.0 ± 0.02% (PAS). During suture, measurements were 98.0 ± 0.008% (MID), 97.0 ± 0.002% (PLA), and 96.0 ± 0.03% (PAS). Post-operative values were 96.0 ± 0.01% (MID), 97.0 ± 0.01% (PLA), and 97.0 ± 0.01% (PAS). The consistent lack of significant differences and maintained high SpO₂ levels indicate that none of the interventions adversely affected respiratory function or oxygen saturation during impacted third molar surgery (Fig. 3D).

Biochemistry

Salivary cortisol

Analysis of salivary cortisol levels revealed significant temporal variations within groups but distinct patterns between groups. For the time factor, both the MID and PAS groups showed reductions in cortisol levels from pre-operative to post-operative periods (MID: 0.53 ± 0.077 to 0.19 ± 0.16; PAS: 0.44 ± 0.11 to 0.18 ± 0.04; P < 0.05). In contrast, the PLA group exhibited no significant pre-post changes (P > 0.05).

In the pre-operative period, MID (0.53 ± 0.07) showed the highest concentration, followed by PAS (0.44 ± 0.11) and PLA (0.32 ± 0.13) (P > 0.05). Post-operatively, significant intergroup differences emerged. The PLA group maintained the highest cortisol concentration (0.42 ± 0.130), which was greater than both the PAS (0.18 ± 0.04; P < 0.05) and MID (0.19 ± 0.16; P < 0.05) groups. No significant difference was found between the MID and PAS groups (P > 0.05), indicating comparable efficacy of midazolam and Passiflora incarnata in reducing biochemical stress responses following surgery (Fig. 4A).

Fig. 4
Fig. 4
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Representative graph of the results obtained from the analysis of salivary biochemical parameters. The parameters evaluated were: (A) salivary cortisol, (B) salivary α-amylase, (C) total salivary protein, and (D) comparison of salivary cortisol between pre- and postoperative periods. Statistically significant differences were observed between the pre- and postoperative periods in the MID and PAS groups for salivary cortisol (p < 0.05). In the intergroup analysis during the postoperative period, both MID and PAS groups showed significant differences compared to the PLA group (p < 0.05). For salivary α-amylase, a significant difference was observed within the PAS group between pre- and postoperative periods, and between PAS and MID groups in the postoperative period (p < 0.05). No statistically significant differences were found for total salivary protein in either intragroup or intergroup comparisons (p > 0.05). Lastly, the comparison of salivary cortisol revealed significant differences among PLA, MID, and PAS groups between the pre- and postoperative periods (p < 0.05).

Salivary amylase

Analysis of salivary α-amylase expression revealed distinct patterns between groups and time points. In the pre-operative period, no statistically significant differences were observed among the PLA (38,194.7 ± 8,649.4 U/L), MID (38,511.1 ± 20,038.2 U/L), and PAS (43,121 ± 29,505.6 U/L) groups (P > 0.05).

Post-operatively, the PAS group exhibited significantly higher α-amylase levels (100,777 ± 43,159.8 U/L) compared to the MID group (47,485.05 ± 21,112.08 U/L; P < 0.05). However, no significant differences were found between PAS and PLA (67,319.4 ± 19,841 U/L) or between MID and PLA (P > 0.05).

Temporal analysis within groups showed a statistically significant increase in α-amylase expression for the PAS group from pre-operative (43,121 ± 29,505.6 U/L) to post-operative (100,777 ± 43,159.8 U/L; P < 0.05). No significant temporal changes were observed for the MID or PLA groups. These results suggest that Passiflora incarnata may uniquely influence sympathetic nervous system activity, as reflected by the pronounced post-operative increase in salivary α-amylase (Fig. 4B).

Total salivary protein

Analysis of total salivary protein levels revealed no statistically significant intergroup differences in either the pre-operative or post-operative periods (P > 0.05 for all comparisons). In the pre-operative phase, values were 1.1762 ± 0.2418 g/L (PLA), 1.1280 ± 0.4229 g/L (MID), and 1.1734 ± 0.3268 g/L (PAS). Similarly, post-operative measurements remained comparable across groups: 1.1340 ± 0.1703 g/L (PLA), 1.3820 ± 0.2138 g/L (MID), and 1.1358 ± 0.4033 g/L (PAS). The consistency in protein concentrations suggests that none of the interventions significantly influenced the overall protein composition of saliva during the perioperative period (Fig. 4C).

Salivary flow

Analysis of salivary flow rates revealed significant temporal reductions within all groups but no intergroup differences at either time point. All three interventions resulted in a statistically significant decrease in salivary flow from the pre-operative to the post-operative period (P < 0.05 for all groups). Specifically, the MID group declined from 0.998 ± 0.185 mL/min to 0.423 ± 0.100 mL/min, the PLA group from 0.899 ± 0.117 mL/min to 0.495 ± 0.187 mL/min, and the PAS group from 0.844 ± 0.252 mL/min to 0.45 ± 0.176 mL/min.

No statistically significant differences were observed between groups in the pre-operative period (P > 0.05), indicating comparable baseline flow rates. Similarly, post-operative comparisons showed no significant intergroup differences (P > 0.05), suggesting that none of the treatments—midazolam, placebo, or Passiflora incarnata—differentially mitigated the surgery-induced reduction in salivary secretion. These findings imply that the observed hyposalivation was primarily a consequence of the surgical procedure itself rather than a specific effect of any pharmacological intervention (Fig. 4D).

Questionnaires

CORAH

Analysis of dental anxiety levels using the Corah questionnaire revealed no statistically significant differences among the three groups at either pre-operative or post-operative time points (P > 0.05 for all comparisons). In the pre-operative assessment, scores were 7.13 ± 2.35 (PLA), 6.33 ± 1.98 (MID), and 6.62 ± 2.21 (PAS). Post-operatively, scores remained comparable across groups: 6.66 ± 1.95 (PLA), 5.93 ± 1.86 (MID), and 6.62 ± 2.21 (PAS). The consistency in anxiety scores suggests that none of the interventions—midazolam, Passiflora incarnata, or placebo—produced a differential effect on subjective anxiety levels as measured by this instrument during the perioperative period (Fig. 5A).

Fig. 5
Fig. 5
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Representative graph of the results obtained from the analysis of psychological assessment questionnaires. The instruments applied were: (A) Corah’s Dental Anxiety Scale, (B) anxiety subscale of the Hospital Anxiety and Depression Scale (HAD), (C) depression subscale of the HAD, and (D) Modified Dental Anxiety Scale (MDAS), evaluated during the pre- and postoperative periods. No statistically significant differences were observed between time points or among groups in either intergroup or intragroup comparisons (p > 0.05).

HAD/anxiety

Analysis of anxiety levels using the HAD scale revealed no statistically significant differences among the three groups in either the pre-operative or post-operative assessments (P > 0.05 for all comparisons). In the pre-operative evaluation, scores were 6.2 ± 2.93 (PLA), 6.2 ± 2.25 (MID), and 6.25 ± 1.80 (PAS). Post-operatively, scores remained consistent across groups: 5.73 ± 2.43 (PLA), 6.13 ± 2.53 (MID), and 6.31 ± 1.92 (PAS). The absence of significant intergroup differences at both time points indicates that none of the interventions—midazolam, Passiflora incarnata, or placebo—produced a measurable differential effect on anxiety levels as quantified by the HAD scale during the perioperative period (Fig. 5B).

HAD/depression

Analysis of depression scores using the HAD scale revealed no statistically significant intergroup differences in either the pre-operative or post-operative periods (P > 0.05 for all comparisons). In the pre-operative assessment, scores were 2.6 ± 1.84 (PLA), 3.86 ± 3.99 (MID), and 3.06 ± 2.14 (PAS). Post-operatively, scores remained comparable across groups: 2.40 ± 1.54 (PLA), 4.06 ± 3.88 (MID), and 2.75 ± 1.98 (PAS). The absence of significant differences suggests that none of the interventions—midazolam, Passiflora incarnata, or placebo—produced a measurable effect on depression scores during the perioperative period of impacted third molar surgery (Fig. 5C).

MDAS

Analysis of dental anxiety levels using the MDAS revealed no statistically significant differences among the three groups in either the pre-operative or post-operative assessments (P > 0.05 for all comparisons). In the pre-operative evaluation, scores were 10.0 ± 3.10 (PLA), 9.53 ± 2.54 (MID), and 9.87 ± 2.26 (PAS). Post-operatively, scores remained consistent across groups: 9.6 ± 2.18 (PLA), 9.46 ± 2.54 (MID), and 9.75 ± 2.21 (PAS). The absence of significant intergroup differences at both time points indicates that none of the interventions—midazolam, Passiflora incarnata, or placebo—produced a differential effect on dental-specific anxiety levels as measured by the MDAS questionnaire during the perioperative period (Fig. 5D).

Patient experience

In the present study, no direct observation of the patients’ surgical experience was conducted; however, it was informally reported that the participants underwent a satisfactory and comfortable clinical process during the impacted impacted third molar extraction procedure, regardless of the treatment administered placebo (PLA), Midazolam (MID), or Passiflora incarnata (PAS). No significant discomfort or perceived differences among the groups were observed, suggesting that all participants felt adequately supported and received humane, high-quality care.

Discussion

Dental anxiety is a common challenge for the dental surgeon. It can be identified in the practice through the observation of changes in the patient’s physiological signs, such as an increased heart rate, higher blood pressure, tremors and difficulty in breathing Gordon D, Heimberg RG, Tellez M et al.; Schou L36,37. The present study has shown that Passiflora incarnata has characteristics similar to Midazolam, without the latter’s well known side effects.

Midazolam has been increasingly used to control anxiety and for preoperative sedation. Despite its advantages, being a rapid onset of action, short duration and low toxicity, it can produce cardiorespiratory instability in case of an overdose, in addition to the risk interaction with other medications, intolerance, nausea and anterograde amnesia Conway A, Rolley J, Sutherland JR et al.; Ong CKS, Seymour RA, Tan JMH; Ong CKS, Seymour RA, Tan JMH; Üstün Y, Gündüz M, Erdoğan Ö et al.38,39,40, which were reported by most patients participating in this study. In contrast, none of the patients who received Passiflora presented amnesia, testament to its low or non-existent interference in memory.

Regarding the expression of biochemical parameters, Steer M and Fromm D41 observed that cortisol secretion in patients undergoing oral surgeries is mainly related to pain during the surgical procedure. Banks P42 showed significant increases in cortisol in the postoperative period. Furthermore Hempenstall PD, Campbell JPS, Bajurnow AT et al.43, corroborating the aforementioned studies, found that cortisol levels remained elevated for approximately 7 h after oral surgery. The study by Jerjes W, Jerjes WK, Swinson B et al.2, in which positive (Midazolam) and negative (Placebo) control were used, showed that the Placebo group had the highest cortisol levels, whereas Midazolam had the lowest values. These findings are in line with the present study, since the group that received the placebo was the one that showed the highest values in the postoperative period. However, when comparing to the Midazolam and Passiflora group, this increase did not occur, which indicates that the administered drugs were able to control salivary cortisol levels.

Salivary amylase is an important marker of stress in saliva, due to changes that occur when the sympathetic nervous system is activated in response to acute stress conditions Robles TF, Sharma R, Park KS et al.44. Moreover, higher expressions of salivary amylase were correlated with the inflammatory response after third molar extraction, notably in patients with greater postoperative pain complaints than those that did not report pain Gutiérrez-Corrales A, Campano-Cuevas E, Castillo-Dalí G et al.45. Supporting the findings of the present study, Robles et al. and Gutiérrez-Corrales et al.45 Also reported a significant increase in salivary amylase in response to acute stress conditions. In the present study, a significant increase in salivary amylase was observed in the placebo group when comparing the pre- and postoperative periods. In the postoperative assessment, a statistically significant difference was found between the Midazolam and Placebo groups, indicating that the placebo was not effective in controlling the elevation of salivary amylase related to surgical stress, thereby highlighting the positive effects of Midazolam in stress modulation. No significant difference was observed between the Passiflora and Placebo groups in salivary amylase levels, suggesting a similar response to surgical stress. Additionally, when comparing the Passiflora and Midazolam groups in the postoperative period, salivary cortisol levels were higher in the Passiflora group, suggesting a comparatively greater reduction of biochemical stress markers with Midazolam.With regard to total protein, no statistically significant differences were observed between the groups for any of the periods. To understand the results, it is necessary to note that the sympathetic and parasympathetic nervous systems act differently on salivary production Keremi B, Beck A, Fabian TK et al.46. What can be conjectured with the observations is that when a patient is on edge from the moment the surgery is scheduled, the action of the parasympathetic system leads to a significant increase in protein production both pre- and postoperatively, resulting in a state of chronic stress.

When evaluating salivary flow, a significant decrease was observed between groups in the pre- and postoperative periods. Although it is well established that elevated stress levels tend to reduce salivary flow, the findings from salivary cortisol and amylase analyses in the present study do not fully support this relationship. It was expected that, with the reduction of biochemical stress markers—amylase and cortisol—salivary flow would increase; however, this was not observed. Bulthuis MS, Jan Jager DH, Brand HS.47 emphasized that chronic stress can persistently reduce salivary flow. Therefore, based on these results, it can be inferred that patients undergoing elective surgeries experience high preoperative stress, which significantly alters their physiology. Although stress levels decreased during surgery, the prior emotional burden may have become chronic, hindering the restoration of normal salivary flow.

Furthermore, the cardiorespiratory rate is an important parameter for assessing anxiety, which may be significantly elevated during anxiety attacks and hyperventilation. With regard to dental procedures, smaller oscillations are expected than those that occur in a state of crisis Dantas L, de Oliveira-Ribeiro A, de Almeida-Souza L et al.28 pointed to an increase in HR in the earliest periods during tooth extraction, lasting up to suturing. In the present study, a slight increase in HR was observed only during the anesthesia period in all groups, with no significant intergroup differences for any of the surgical periods. This fact shows that different medications had a similar effect on HR during third molar extraction. Some studies have pointed out that even placebo can generate a satisfactory result, due to its effect at a psychological level, with anxiety control sometimes better than expected48,49,50.

Similarly, blood pressure may be high at the beginning of the procedure, especially in patients with a dental phobia. It is important to note that no patients with a history of high blood pressure were included in this study. Systolic BP did not show any intergroup changes. Regarding diastolic BP, there was a statistically significant difference at the time of anesthesia, albeit not far removed from baseline values crisis Dantas L, de Oliveira-Ribeiro A, de Almeida-Souza L et al.28 also observed specific changes in diastolic BP, more specifically an increase in values at the time of extraction, followed by a drop at the time of suturing. Therefore, it is possible to assume that diastolic pressure is more sensitive to small changes at moments of greater emotional stress for the patient like anesthesia, due to the presence of a needle and tissue perforation, as well as during tooth extraction, when the exerted pressure tends to generate anxiety in patients.

Another common concern in the dental practice when using benzodiazepines to control anxiety is the risk of mild respiratory depression. Respiratory depression is more worrying when the dosage aims at a moderate sedation of the patient with values in excess of 7.5 mg, the dosage used in this study. Notwithstanding, even at lower dosages used to control anxiety, the dentist should always have an oximeter accompanying the patient during the procedure, given that some patients may be more sensitive to the effect of the medication and have a significant drop in blood oxygen saturation. No statistically significant differences were found in the values obtained through oximetry. As evidenced in previous studies de Moares MB, Barbier WS, Raldi FV et al.; Hari Keerthy P, Balakrishna R, Srungeri KM et al.; Miyasaka LS, Atallah ÁN, Soares B. 13,51,52, group comparisons demonstrated that Midazolam, Passiflora, and placebo did not result in significant changes in peripheral oxygen saturation (SpO₂). Although Midazolam is considered safe when used appropriately Hari Keerthy P, Balakrishna R, Srungeri KM et al.51, Passiflora may represent a valuable alternative due to its lower potential for adverse effects.

Clinical studies have not identified any health hazards related to the use of Passiflora Incarnata Miyasaka LS, Atallah ÁN, Soares B.; Movafegh A, Alizadeh R, Hajimohamadi F et al.; Jawna‐Zboińska K, Blecharz‐Klin K, Joniec‐Maciejak I et al.52,53,54 conducted a study with prolonged use of this medication in rats, which demonstrated a decrease in stress levels and, consequently, an increase in the animals’ motivation to act and an improvement in motor activity. Their demonstration of Passiflora’s cumulative effect constitutes a limiting factor for the present study, as the use of this medication for long periods is not viable for elective surgical procedures that require scheduling in a short period Dantas L, de Oliveira-Ribeiro A, de Almeida-Souza L et al.28 used an experimental model of lower third molar extraction. Participants received 15 mg of midazolam or 260 mg of Passiflora Incarnata, 30 min before the surgical procedure. They concluded that the anxiolytic action of the two medications was similar, but that some of the participants who received Midazolam reported not remembering anything. This was not the case with the patients who received Passiflora. Drowsiness was also reported by 82.5% of those who received Midazolam, against 50% in the Passiflora group.

A study by Azimaraghi O, Yousefshahi F, Khatavi F et al. 55 aimed to compare the efficacy of Passiflora incarnata and Oxazepam in reducing preoperative anxiety. Patients who received Passiflora tablets had lower levels of anxiety than those who were given Oxazepam. The effect on psychomotor functions was similar, as was the recovery time. The authors concluded that the herbal medicine was more effective in reducing preoperative anxiety, and that it can be included in the treatment of anxiety in children and adolescents. It should be noted that there is no specific protocol for the use of Passiflora. For example, Dantas L, de Oliveira-Ribeiro A, de Almeida-Souza L et al.28, used the lowest commercially available dose of 260 mg, while Movafegh A, Alizadeh R, Hajimohamadi F et al.53, Christoffoli MT56, and da Cunha RS, Amorim KS, Gercina AC et al.57 administered a 500 mg dose. Despite these differences in dosing, all these studies consistently demonstrated the effectiveness of Passiflora, with some results comparable to those obtained with Midazolam. These direct findings from Azimaraghi O, Yousefshahi F, Khatavi F et al.55 and the other referenced studies strongly support the results observed in the present study.

The subjective assessment of anxiety using the CORAH, HAD, and MDAS questionnaires may not have fully captured the patients’ stress levels, as no statistically significant differences were observed between the groups from the pre- to postoperative periods. This suggests that the absence of a specific questionnaire designed for surgical procedures could be a contributing factor to the lack of statistically significant intergroup differences.

Based on the observed results, it is concluded that the use of Passiflora incarnata represents a favorable alternative for patients with contraindications to benzodiazepines. The stress-reducing effect, as evidenced by the analysis of salivary cortisol levels, was statistically equivalent to that of Midazolam. Crucially, this outcome was achieved without the adverse effects associated with benzodiazepine medication, suggesting a superior safety profile for passiflora in the context of oral surgery.

This study is subject to some limitations. The potential contamination of postoperative saliva samples with blood may have influenced the analysis of stress biomarkers. Furthermore, the dosage of Passiflora incarnata used was based on preliminary protocols, as there is no established consensus regarding the ideal posology for surgical procedures. Although the calculated statistical power was achieved, a larger patient cohort would strengthen the statistical significance of the findings. Finally, the influence of the placebo effect, while inherent to the study design, remains a potential bias that must be considered when interpreting the results.

Against this background, it is recommended that future studies investigate higher doses of Passiflora to reinforce its possibilities, safety, efficacy and advantages over to other medications like nitrous oxide. Another interesting approach would be the association of sedative agents with preemptive drugs, since stress is directly associated with the release of inflammatory factors and, consequently, the exacerbation of postoperative pain. Furthermore, it is important to consider the use of saliva as a biomarker of stress and anxiety levels in different dental procedures, as it is an easy and non-invasive method, thus expanding the range of possibilities for oral surgeries.

Despite the limited number of scientific studies evaluating the anxiolytic activity of Passiflora incarnata in third molar extraction procedures, the available evidence and the findings of this clinical study suggest that Passiflora incarnata may offer anxiolytic effects comparable to those of Midazolam, with good tolerability and no observed adverse effects. Therefore, it could represent a promising pharmacological option for managing anxiety during dental treatment.

Conclusions

Based on the physiological and biochemical salivary analyses, it can be concluded that Passiflora Incarnata at a preoperative dosage of 260 mg has an anxiolytic effect similar to Midazolam, without presenting any side effects. Therefore, it is both safe and effective in reducing stress and anxiety in adult patients undergoing third molar extraction.