The In Vitro Evaluation of Acaricide Paeonol against Human Demodex (Acari: Demodicidae)

Demodex, a genus of the family Demodicidae, sub-order Euacarophora, order Acariformes, and Arachnid, are a permanent ectoparasite of humans and mammals. Depending on the host and its morphological characteristics, more than 100 species or subspecies can live on humans, dogs, sheep, cattle, pigs, cats, mice, bats, pandas, and other mammals (Foley et al., 2021; Smith et al., 2022; Wei et al., 2023). Human Demodex, including Demodex brevis and Demodex folliculorum, inhabit the sebaceous glands and follicles of eyelashes and other body hairs, such as the nose, ears, beard, and eyebrows. Under normal conditions, D. brevis is more broadly dispersed throughout the body than D. folliculorum, despite the latter being more visible (Norn, 1982; Elston, 2010; Palopoli et al., 2015). D. folliculorum and D. brevis survive by accessing food in the hair follicle and sebaceous gland cells through their needle-like mouths (Paichitrojjana, 2022). Human Demodex species are the most complex and commensal residents, but they can lead to some diseases and clinical symptoms termed demodicosis, such as decreased host immunity, sebaceous hyperplasia, and erythematotelangiectatic rosacea (Elston and Elston, 2014; Forton and De Maertelaer, 2021; Clanner-Engelshofen et al., 2022). Hence, the prevention and control of demodicosis are beneficial for physical and mental health of patients with demodicosis. Currently, the treatment of demodicosis mainly depends on chemical agents, including metronidazole, ivermectin, permethrin, benzoyl benzoate, crotamiton, lindane, and sulfur (Jacob et al., 2019; Paichitrojjana, 2022; Chudzicka-Strugala et al., 2023). Nevertheless, several studies have confirmed that these medicines could induce additional adverse drug reactions (Jacob et al., 2019).

Paeonol (2ʹ-hydroxy-4ʹ-methoxyacetopheone) is the principle bioactive compound isolated from the root bark of moutan cortex (Paeonia suffruticosa Andrew) and is known for its natural bioactive phenol with antioxidant properties (Gyawali et al., 2019; Zhang et al., 2019; Adki and Kulkarni, 2020; Wang et al., 2020; Ding et al., 2023). Paeonol has prolonged clinical use history in morden China, and its dosage forms date back to as early as the 1970s (Zhang et al., 2019). The pharmacological activity of paeonol was first recorded in traditional Chinese medicine 50 years ago (Adki and Kulkarni, 2020). Previous studies have shown that paeonol had been extensively used because of its great pharmacological potential as an anti-inflammatory, anti-fungal, anti-viral, and insecticidal compound (Xu et al., 2003; Adki and Kulkarni, 2020; Lv et al., 2020; Li et al., 2021; Tang et al., 2022). Some studies have reported that paeonol is active against multiple species of mites, such as Dermatophagoides farina, Dermatophagoides pteronyssinus, Tyrophagus putrescentiae, and Aleuroglyphus ovatus (Kim et al., 2004; Tak et al., 2006; Zou et al., 2023a, 2023b). However, it remains unclear whether paeonol has an acaricidal effect on Demodex. The present study was performed to evaluate the acaricidal efficacy of paeonol against human Demodex.

Live mites dwelling in human sebaceous glands and hair follicles were obtained anonymously through the cellophane tape method. Transparent glue was pasted on the nose, nasal groove, forehead, zygoma, and chin of Demodex-positive patients until the next day, after being instructed to clean their face with tap water before sleeping. Next, the tape was removed and placed on the center of a new clean slide. Paeonol and paraffin oil were purchased from Shanghai McLean Biochemical Technology Co., Ltd.

Paeonol was diluted to the following concentrations with paraffin oil at 50 °C: 60, 50, 40, 30, 20, and 10 mg/mL. The living adult mites on the slides with activity intensity were observed, classified, and screened by light microscopy (Nikon, Tokyo, Japan) at 25–27 °C. Subsequently, each selected mite was placed on an independent slide. And, the drug solution to be tested was added to uniformly cover the mite body surface, and the slides were placed in a wet box with 60–70% relative humidity at room temperature. The survival and activity of mites were observed under the microscope and recorded at fixed periods. The mites were considered dead when the body pincers and tarsi were motionless for 1 min and remained unmovable when stimulated with an anatomical needle. The survival time of the mites was recorded. Negative control individuals were also evaluated by treatment with paraffin oil. In the 60, 50, 40, 30, 20 and 10 mg/ml groups, the numbers of D. folliculorum tested were 30, 31, 31, 33, 32 and 30, respectively; in the same concentration groups, the numbers of D. brevis were 27, 28, 27, 28, and 28, severally. In the negative control groups, the numbers of D. folliculorum and D. brevis were 10 and 13.

All statistical analyses were performed using IBM-SPSS software version 26 (IBM, Armonk, NY, USA). The primary statistical methods included the chi-square test and linear regression analysis.

The mites were observed under a light microscope (Nikon, Tokyo, Japan) (Fig. 1). The initial state of the mites revealed that their holonomic bodies were active, with five or six tarsi; they also moved 6–8 times in a min (Fig. 2A, D). Following exposure to the paeonol solution, the bodies of both D. folliculorum and D. brevis significantly contracted and twisted; the body condition (more than six tarsi moved for more than 10 times in 1 min) was more strenuous than the initial activity state (Fig. 2B, E). After considerable struggle, their bodies gradually returned to their original appearance. The movement of the mites was delayed, podosoma and gnathosoma were expanded, opisthosoma was shortened, and the bodies of the mites became slightly more transparent than their primal form on death. There was no sign of movement in their pincers and tarsi for 1 min, even these parts were stimulated with a skinny anatomical needle, particularly in the 60, 50, and 40 mg/mL concentration groups (Fig. 2C, F). In the final stage of the 8-h observation period, the mites showed weak motor performance; only one leg moved 1–2 times in 1 min in response to the abovementioned stimulation, and the bodies of the mites did not change in the 30 mg/mL concentration group. The groups treated with paeonol at

10 or 20 mg/mL concentrations showed relatively vigorous movements without any needle, with the movement of two or three tarsi 3–5 times per min.

As shown in Table I no recorded deaths occurred in the pure paraffin oil negative control group. However, in the test groups, mite death was observed in the 60, 50, 40, 30, 20, and 10 mg/mL paeonol solutions containing paraffin oil for 8 h. Hence, the average dead time at 60, 50, and 40 mg/mL paeonol concentrations was 118, 142.2, and 240 min, respectively; furthermore, the average dead time was more than 480 min for 30, 20, and 10 mg/mL paeonol concentrations. The mortality rates in 8 h were 100%, 100%, 90.3%, 6.0%, 3.1%, and 3.3%, respectively. The results showed that with decreasing drug concentrations, the acaricidal effect faded over time, and the death time increased incrementally. No significant difference was observed between the 30 mg/mL group and the negative control group, the 20 mg/mL group and the negative control group, and the 10 mg/mL group and the negative control group (χ2 = 0.001, P > 0.05; χ2 = 0.001, P > 0.05; χ2 = 0.001, P > 0.05) based on the statistical analysis of the 8-h mortality period. A significant difference in mortality was noted between the 60, 50, and 40 mg/mL groups and the negative control group (χ2 = 34.844, P < 0.05; χ2 = 35.757, P < 0.05; χ2 = 24.468, P < 0.05). Therefore, the 40 mg/mL dose was the lowest effective concentration of paeonol on D. folliculorum in this study in 8 h.

Table I shows that the average dead time was 98.6, 123.4, and 181 min for the 60, 50, and 40 mg/mL paeonol concentrations, respectively. Additionally, more than 480 min was required to kill mites at 30, 20, and 10 mg/mL concentrations. In the negative controls, all mites remained active. The mortality rates in 8 h were 100%, 100%, 92.6%, 17.8%, 7.1%, and 7.1% for 60, 50, 40, 30, 20 and 10 mg/mL concentrations, respectively. No apparent difference in the mortality rate was noted between the 30, 20, and 10 mg/mL groups and the negative control group (χ2 = 1.239, P > 0.05; χ2 = 0.440, P > 0.05; χ2 = 0.440, P > 0.05, respectively). However, the 60, 50, and 40 mg/mL groups showed a significant difference when compared with the negative control group (χ2 = 35.571, P < 0.05; χ2 = 36.512, P < 0.05; χ2 = 28.270, P < 0.05, respectively). Thus, the 40 mg/mL dose was the lowest effective concentration of paeonol against D. brevis in 8 h.

Table I and Figure 3 suggest that the average death time for D. brevis (98.6, 123.4, and 181 mins) was less than that of D. folliculorum (118.2, 142.2, and 240 min) in 60, 50, and 40 mg/mL concentration groups, respectively. Thus, an apparent difference was noted between D. brevis and D. folliculorum in the average death time at the same concentration groups. D. folliculorum (81, 72, 145, 365, 422, and 436 mins) showed a longer survival time than D. brevis (59, 63, 96, 322, 391, and 391) at the same concentration groups of paeonol. The death rates of D. folliculorum in 8 h were 100%, 100%, 90.3%, 6.0%, 3.1%, and 3.3% for 60, 50, 40, 30, 20, and 10 mg/mL paeonol concentrations, respectively; the death rates of D. brevis were 100%, 100%, 92.6%, 17.8%, 7.1%, and 7.1% for 60, 50, 40, 30, 20, and 10 mg/mL paeonol concentrations, respectively. Additionally, paeonol could kill more D. brevis than D. folliculorum, though higher drug concentrations resulted in faster death for both mite species. The average death time decreased with the increasing concentration of paeonol for both mite species. Thus, paeonol exhibited superior acaricidal activity against D. brevis than against D. folliculorum in terms of average death time, survival time, and mortality rate in 8 h.

Paeonol, with better therapeutic effects on pigmentation, psoriasis, eczema, and itchy skin, can bind to tyrosinase and inhibit the activity of enzymes to achieve whitening function (Wang et al., 2020; Min et al., 2023). Holmes (2013) found that bacteria in Demodex could be involved in the development of rosacea and other facial dermatoses (Lazaridou et al., 2011; Jarmuda et al., 2012). Other studies have shown that symptoms were relieved after antibacterial treatment (Hsu et al., 2009; Martinez-Diaz et al., 2012; Guerrero-González et al., 2014). Zhao (2017) indicated that Staphylococcus and Sphingomonas might be associated with the development of facial dermatoses by the denaturing gradient gel electrophoresis (DGGE) technique (Zhao et al., 2017). Zeng et al. (2022) demonstrated that paeonol exhibits an antibacterial effect on Staphylococcus aureus (Zeng et al., 2022). Thus, we can reasonably expand the real-world applications of paeonol on skin care products. Heczko et al. (2023) reported that the mites were considered dead when all the rotary movements of Demodex appendages ceased (Fig. 2G, H). Clanner-Engelshofen (2022) proposed that the immotile mites with symmetrically spaced legs were potentially dead owing to muscular flaccidity (Clanner-Engelshofen et al., 2020). In this study, we observed the body pincers and claw for 1 min and then stimulated the mite with an anatomical needle; if it remained motionless, we considered the mite to be dead (Clanner-Engelshofen et al., 2020). We also discovered some coelom and/or internal hydroskeleton in live D. folliculorum (Fig. 2I). The solid hydroskeleton may help the body to move by decreasing the contact surface to reduce friction when walking (Clanner-Engelshofen et al., 2020).

Although the 30, 20, and 10 mg/mL groups showed no significant difference with the negative control group in terms of the mortality rate in 8 h of treatment, the vitalities of mites were notably different at 8 h. The majority of mites exposed to the 30 mg/mL concentration of paeonol did not die but were carotic; only one leg moved with stimulation at the final stage of the 8-h in this research. However, the mites in the 20 or 10 mg/mL group actively moved even without needle stimulation at the end of 8 h. Therefore, a significant difference was noted between the 30 mg/mL group and the negative control group when the drug application time was prolonged. A favorable correlation was noted between time and concentration and the total number of deceased human Demodex mites. The number of dead mites in 8 h increased as paeonol concentration increased, and the mortality rate reduced as paeonol concentration decreased. This finding was comparable to previous reports (Zhao et al., 2006; Du et al., 2021).

In this study, paeonol had a distinct killing effect on two types of human Demodex mites (D. folliculorum and D. brevis). The movements were delayed, podosoma and gnathosoma expansion occurred, even shortening arose in the opisthosoma, and the bodies became slightly more transparent than primal form when death. The killing time was lengthened with decreasing concentration, thus showing an evident dependence on concentration. According to the experimental findings, paeonol had a minimum effective dose of 40 mg/ml for killing both D. folliculorum and D. brevis, with paeonol having a higher killing effect on D. brevis than D. folliculorum during the 8-h period.

The authors are grateful to the Demodex-positive patients for providing a large number of mites to carry out the work.

This work was supported by grants from the Academic Aid Program for Top-notch Talents in Provincial Universities (gxbjZD2020071), Wuhu Key Research and Development Program of China (2021yf391) and Young and Middle-aged Research Fund of Wannan Medical College (WK202216).

The study was approved by Wannan Medical college (Wuhu, China), and all participants signed a written informed consent forms.

The authors confirm that the data supporting the findings of this study are available within the article.

The authors have declared that no generative AI or AI-assisted technologies were used to create this manuscript.

The authors have declared no conflict of interest.