Modulation of TRPV1 function by Citrus reticulata (tangerine) fruit extract for the treatment of sensitive skin

Sensitive skin (SS) is a clinical syndrome defined by the occurrence of unpleasant sensations (such as stinging, burning, pain, pruritus, and tingling) in response to stimuli that normally should not provoke them. According to growing evidence, transient receptor potential vanilloid subtype 1 (TRPV1) has elevated expression in individuals with SS and is linked with the severity of SS symptoms. However, its pathogenesis is still unknown.

genesis is still unknown.
Objective: Herein, Citrus reticulata (Tangerine) fruit extract (CR) was obtained and examined for its effect on SS with a focus on TRPV1 stimulation and expression.

Methods: A recombinant hTRPV1 over-expression cell line (HaCaT-TRPV1-OE cell)
was constructed to screen substances and extracts from several plants. Intracellular calcium mobilization was monitored by Flexstation 3 and a fluorescence microscope using Fluo 8 AM fluorophore. Next, immunofluorescence was used to detect the TRPV1 expression under different stimulants treated for 24 h. To investigate the relief and increased tolerance of CR to lactic acid-induced skin discomfort, clinical tests were carried out on the nasolabial folds or cheek areas.
Results: According to the obtained results, compared to HaCaT cells, HaCaT-TRPV1-OE cells showed a higher expression of TRPV1. Neuronal hyperresponsiveness in SS triggered by capsaicin (CAP), lactic acid, phenoxyethanol or nicotinamide may be through activation of TRPV1 and increased TRPV1 expression. CAP activates TRPV1 in HaCaT-TRPV1-OE cells, and more than 100 plants or chemicals were tested for their inhibitory effects before being screened for CR. CR (1%-4%) inhibited TRPV1 activation induced by CAP or phenoxyethanol or nicotinamide. Meanwhile, CR (0.25%) suppressed TRPV1 protein expression induced by phenoxyethanol or lactic acid. In vivo results showed that CR not only instantly relieved lactic acid-induced skin discomfort under 5 min but also enhanced skin tolerance to lactic acid after 7 days of continuous use.
Conclusions: Topical application of CR showed an instant and long-lasting improvement in SS by modulating the activation and expression of TRPV1. Moreover, it has been suggested that CR might act as a TRPV1 inhibitor to reduce skin irritation or sensitivity.

| INTRODUC TI ON
Sensitive skin (SS) is a syndrome that involves unpleasant sensations such as stinging, burning, pain, pruritus, and tingling in response to stimuli that normally should not provoke them. These unpleasant sensations cannot be explained by lesions linked to any skin disease. The skin can be normal or accompanied by erythema. The International Forum for the Study of Itch (IFSI) states that SS can affect any part of the body, but it is more common on the face. 1 According to epidemiological research, sensitive skin affects up to 70% of women and 60% of men. 2 The patient's life quality is negatively impacted by the disease, which is upsetting.
The pathogenesis of SS is still under investigation. However, multiple recent investigations have identified discrepancies between the normal dermis and SS in terms of the epidermal barrier function, as well as vascular and neuronal hyper-reactivity in the normal dermis versus SS. 3,4 In addition, several studies have reported a correlation between the expression of transient receptor potential vanilloid subtype 1 (TRPV1) and the severity of SS symptoms. 5,6 TRPV1, a non-selective cation channel, is widely expressed in nociceptive neurons (C-and Aδfibers) and non-neuronal cells, such as keratinocytes, fibroblasts, and endothelial cells. 7 It can be activated by various stimuli, including heat (>43°C), low pH (<5.9), capsaicin (CAP), phenoxyethanol, and a growing list of endogenous and exogenous compounds. Activation of TRPV1 is followed by increased permeability/influx of Na + and Ca 2+ activating linked signal transduction pathways exerting regulatory effects. 7 Phytochemical studies showed that Citrus reticulata (Tangerine) fruit extract (CR) is rich in flavonoids and alkaloids 8 which are responsible for CR's analgesic properties. 9 Among them, naringenin and hesperetin are the most important flavanones that can potently and selectively block transient receptor potential cation channel subfamily M member 3. 10 However, the regulatory mechanism of CR on pain concerning cutaneous discomfort remains elusive.
This research aims to identify a TRPV1 inhibitor with an abiological mechanism that might be used as a cosmetic active ingredient to treat SS. Against TRPV1, a high-throughput screening model (HaCaT-TRPV1-OE cell) was designed. Several cosmetic compounds with the potential to cause irritation were chosen to determine whether they activate TRPV1 and can be inhibited by TRPV1 inhibitors. We found that besides CAP, phenoxyethanol, nicotinamide, and lactic acid may be associated with the modulation of the TRPV1 receptor and CR relieves SS by regulating the activation and expression of TRPV1.

| Preparationof Citrus reticulata fruit extract (CR)
Briefly, the selected fruits (0.5 kg) were extracted with water (5.0 kg) for 1.5 h at 90°C. The extract was enzymatically hydrolyzed with pectinase for 1 h at 45°C. Next, the extract was heated at 90°C for 30 min to eliminate proteins and then filtered with a polypropylene membrane (0.8 μm pore size). The filtrate (about 3.8 kg) was subjected to column chromatography over D101 macroporous resin (7.5 × 30 cm) and then eluted with water (1.5 kg), 30% butylene glycol (1.5 kg), and 40% butylene glycol (2 kg). To obtain CR, the pH of the 60% butylene glycol fraction was adjusted to 4.0-4.5 and UV absorption spectroscopy was used to identify the active components in CR. Citrus reticulata (tangerine) fruit extract, lactic acid, sensitive skin, skin discomfort, TRPV1 24 hours. A concentration of CR ranging from 0.03% to 0.25% was added to the cells alongin conjunction with 5 μM Fluo-8 AM dye for 30 min, and then cells were treated with either 5 μM CAP or 0.4% phenoxyethanol for 10 min before being rinsed. A fluorescence microscope was used to observe the fluorescence of the sample (excitation 485 nm, emission 525 nm).

| Samples
Formulas for CR-containing lotions with concentrations of 0.5% and 1% are shown in Table 1. The base formula without CR was considered a placebo group (negative control). Before in vivo study, samples were tested for adverse skin reactions using a 24 h skin patch test.

| Subjects and screening procedures
The selection process for SS participants was conducted in two stages. To begin, people aged 20-45 who believed they had SS filled out a questionnaire. 12 Pregnant women, people with known or suspected malignancies, those with scars or wounds at the testing site, and those with cutaneous disorders like acne, atopic dermatitis, and rosacea were not permitted to take part. A lactic acid sting test was also administered, and participants were selected if they had a total sting score of more than 3 at both 2.5 and 5 min. The lactic acid sting test was carried out exactly as described elsewhere. 13 To apply lactic acid to the nasolabial folds or cheeks, a cotton swab was dipped in a 10% lactic acid solution. Discomfort on a 5-point scale was recorded within 5 min of applying lactic acid to the skin (0, no discomfort; 1, mild discomfort; 2, moderate discomfort; 3, severe discomfort; 4, extreme discomfort).

| Instant skin soothing test
The instant skin soothing test included 28 participants (10 males and 18 females) with SS and an average age of 29 years. Initially, a 10% lactic acid solution was applied to the nasolabial folds or cheeks using a cotton swab; the sting score was recorded 2.5 min after application using the 5-point scale shown above; this score was also used as the sting score prior to the application of 1% CR (0 min).
After this, 1% CR was applied to the same areas of each subject's cheeks or nasolabial folds at a dose of 20 μl/cm 2 immediately, and skin discomfort scores were recorded at 2.5 and 5 min after the application of 1% CR using a 5-point scale (as indicated above).

| Long-term skin tolerance test
Twenty-four subjects (8 males and 16 females) with SS and an average age of 28 years were included for long-term lactate tolerance testing. First, on Day 0, we performed the lactic acid stinging test by applying 10% lactic acid solution with a cotton swab to the left and right nasolabial folds or cheeks of the subjects, respectively, and recording the stinging score at 1 and 5 min after application of 10% lactic acid on a 5-point scale (as indicated above), before washing the face with warm water.
Step 2 involved a half-face control application of a sample: half of the faces received base (placebo) and the other half received 0.5% CR at a dose of 20 μl/cm 2 every morning and night for seven days. Third, we repeated the lactic acid stinging test in the afternoon of day 7 to get stinging scores again, as we did in steps 1 and 2.

| Statistical analysis
Data were reported as means ± SEM based on at least three experiments. Statistical analyses were performed using GraphPad 5.0 sta- To determine the % of inhibition following formula was used.

| HaCaT-TRPV1-OEcellsasascreeningmodel for SS
According to previous research, TRPV1 expression is elevated in SS patients, and its levels positively correlate with disease severity. 5,6 As shown in Figure

| CRactsasaTRPV1inhibitor
Hundreds of differently processed plant extracts and compounds were screened using CAP-treated HaCaT-TRPV1-OE cells to identify potent and effective inhibitors of TRPV1. Herein, a patented, specialized plant extraction method was used to produce CR. According to the obtained data, CR contained more than 0.6% alkaloids and 0.6% flavonoids.
As shown in Figure 2A, a 1%-4% concentration of CR inhibited not only the 4 μM CAP stimulated calcium influx but also that from 0.4% phenoxyethanol and 5% niacinamide in a dose-dependent manner; the maximum inhibition was >95.0%. To further clarify the CR antagonistic effect in cells against TRPV1 activation, a fluorescence microscope assay was performed to identify the continuous activation of TRPV1. Compared to the control group, both the phenoxyethanol and CAP groups had strong green fluorescence. Figure 2B shows that when CR was treated with phenoxyethanol or CAP at concentrations between 0.06% and 0.25%, the intensity of the fluorescence decreased with the dose. These results suggested that CR can inhibit both transient and continuous activation of TRPV1 receptors induced by phenoxyethanol or CAP.
This study also evaluated the effect of CR on TRPV1 protein expression ( Figure 2C). TRPV1 expression significantly increased when cells were treated for 24 h with a concentration of 0.05% lactic acid and 0.03% phenoxyethanol than the control (red fluorescence).
However, this phenomenon was absent in the 5 μM CAP group.
These data suggested that CAP mainly activates TRPV1, but longterm use of lactic acid and phenoxyethanol not only activates TRPV1 but also upregulates its expression, aggravating SS.
Interestingly, 0.125%-0.25% concentration of CR can significantly inhibit lactic acid or phenoxyethanol-induced upregulation of TRPV1 expression. These results indicated that CR acts as an inhibitor of TRPV1 activation and expression against different stimulants.

| TopicallyappliedCRcouldimprove skin discomfort
Herein, 54 participants satisfied the screening and exclusion criteria and were positive for lactate tingling following a two-step screening approach. Furthermore, 28 and 24 subjects were included in the instant skin soothing effect and long-term skin tolerance effect studies, respectively. In addition, lactic acid sting test carried out during the subject screening process have found that skin discomfort is consistently increased between 2.5 and 5 min after the application of lactic acid, if no effective substance is used to relieve this discomfort and can be prolonged more than 10 min. The results of the instant soothing effect of CR on lactic acid-induced skin discomfort are shown in Figure 3A. Compared to before application, lactic acid-induced skin discomfort was reduced by 58.43% at 2 min and 76.99% at 5 min after 1% CR. The results of the skin tolerance to lactic acid study were shown in Figure 3B. The placebo group (base) did not have a significant relieving effect against lactic acid-induced skin discomfort, whereas, patients' tolerance to lactic acid-increased after 7 days of continuous application of 0.5% CR.
Additionally, the lactic acid induced skin discomfort after CR treatment reduced by 68.00% and 52.29% at 1 and 5 min, respectively, compared to before CR application. In view of the obtained data, CR not only provided immediate relief but also improved the tolerance of SS to lactic acid.

| DISCUSS ION
The diagnosis and assessment of SS mainly involve questionnaire surveys and sensory analysis, such as stinging tests with lactic acid, 14 CAP, and dimethyl sulfoxide (DMSO). 15 However, these strategies are According to a previously reported study, the role of keratinocytes in SS may not be limited to alterations of the epidermal barrier but can also be related to their sensory properties such as pain and itching. 16 Additionally, keratinocytes are considered to provide only  CR was identified from hundreds of samples and evaluated in CAPinduced HaCaT-TRPV1-OE cells. Furthermore, CAP, phenoxyethanol, nicotinamide, and lactic acid were used as TRPV1 activators to explore the underlying mechanism of CR on TRPV1 activation and F I G U R E 3 CR treatment improved lactic acid (10%) induced skin discomfort. (A) Instant skin sting test (n = 28). The skin discomfort was scored after 10% lactic acid induction at 0.0 (before sample application), 2.5, and 5.0 min of the sample application. The relief rate in skin sensitivity to lactic acid was calculated as follows: (Score after -Score before ) × 100%/ Score before , *** 2.5 min or 5 min group vs 0 min group, p < 0.001. (B) Long-term skin tolerance effect (n = 24). Base group: placebo group. The relief rate in skin sensitivity to lactic acid was calculated as follows: (Score 7day -Score 0day ) × 100%/ Score 0day . ** p < 0.01, *** 0 Day group vs 7 Day group, p < 0.001.
expression. Finally, in vivo skin, soothing studies validated the immediate soothing and skin tolerance-enhancing effect of CR on sensitive skin. This study demonstrated that CR alleviates SS by reducing TRPV1 activity and expression.

ACK N OWLED G M ENTS
We would like to thank the ethics committee of Shanghai JAKA Biotech for kindly approving this clinical study and the team of Wei-Qiang Lu from East China Normal University to support our substance screening by providing Flexstation3 instruments.

FU N D I N GI N FO R M ATI O N
This study was funded in full by Shanghai JAKA Biotech Co. Ltd. All authors are employees of Shanghai JAKA Biotech Co. Ltd.

CO N FLI C T O F I NTE R E S T
The authors have no other conflict of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH C A LA PPROVA L
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. Clinical studies have been approved by the ethics committee of Shanghai JAKA Biotech Co. Ltd. on 13th July 2022, with an approval number of JKC-2207-00020.