Guang Chenpi Extract as a Multifunctional Phytotherapeutic: Enhanced Effects with Ergothioneine and Polydeoxyribonucleotide on Redox Homeostasis and Tissue Resilience

Abstract

Background/Objectives: Guang Chenpi, the aged pericarp of Citrus reticulata ‘Chachi’, is a traditional Chinese medicinal food with documented health benefits. This study aimed to systematically evaluate the multifaceted bioactivity of a standardized Guang Chenpi extract (GCE), both alone and in combination with ergothioneine (EGT) and polydeoxyribonucleotide (PDRN), using in vitro and in vivo models. Methods: GCE quality was characterized by LC-MS/MS. Combination regimens of GCE with EGT or PDRN were assessed in UVB-irradiated 3D MelaKutis^® skin tissue for ROS levels, antioxidation defense markers (NNT, GSH-PX1), and melanocyte protein (Pmel17). In zebrafish, GCE was evaluated for toxicity, antioxidant activity, tail fin regeneration, skin barrier protection, melanogenesis inhibition, and expression of collagen (col1a1a, col1a1b, and col1a2) and elastin (elna) genes. Results: In 3D skin models, GCE combined with EGT or PDRN significantly enhanced antioxidant defenses (NNT increased by 113–186%; GSH-PX1 by 173–231%), reduced ROS by 46.27–57.76%, and decreased melanocyte protein (Pmel17) by 23.44–44.27%. In zebrafish, GCE showed low toxicity (≤0.63 mg/mL) and exhibited dose-dependent antioxidant activity (ROS reduction: 27.57–61.85%), enhanced tail fin regeneration (11.35–27.84%), and strengthened skin barrier function (65.20–89.32% protection). GCE also upregulated collagen and elastin gene expression, improved blood circulation, and suppressed melanogenesis. Conclusions: GCE is a promising multifunctional natural ingredient with significant antioxidant, regenerative, and skin-protective properties. Its combination with EGT or PDRN results in enhanced protective effects against UVB-induced skin damage, supporting its potential use in advanced pharmaceutical and cosmeceutical formulations.

1. Introduction

Guang Chenpi, the dried pericarp of Citrus reticulata ‘Chachi’, represents one of the most distinguished traditional Chinese medicines with both medicinal and edible applications. Primarily produced in Guangdong Province, China, it accounts for approximately 70% of the total Chenpi production nationwide [1]. Officially recorded in the Chinese Pharmacopoeia [2], Guang Chenpi has been extensively used for thousands of years in clinical practice for regulating qi, treating nausea, vomiting, cough with phlegm, dampness, and strengthening spleen function [3,4]. Its widespread use as a medicinal food extends throughout Southern China and other Asian countries [5], underscoring its significant cultural and therapeutic value.

Modern pharmacological investigations have progressively unveiled the multifaceted bioactivities of Guang Chenpi. Substantial evidence demonstrates its anti-cancer properties [6]; cardiovascular protective effects against myocardial infarction, heart failure, and cardiac hypertrophy [7]; regulation of blood glucose and lipids [8]; alleviation of chronic lung diseases [9]; anti-asthmatic effects [10]; gut microbiota modulation [11]; immune enhancement; neuroprotection [12]; anti-diabetes [13]; as well as antibacterial, antiviral [3,5], and antioxidant activities [4]. These diverse pharmacological effects are primarily attributed to its rich composition of bioactive compounds, particularly flavonoids, which include hesperidin, nobiletin, and tangeretin.

The quality and efficacy of Guang Chenpi are intrinsically linked to its traditional processing method, which mandates a minimum three-year aging period. This process, known as “Chen Jiu Zhe Liang” in traditional Chinese medicine, emphasizes that longer aging results in superior quality [14]. The aging process constitutes a sophisticated microbial fermentation that enriches pharmacologically active ingredients, especially flavonoids, followed by phenolic acids [1,14,15], terpenes, alkaloids, essential oils, and polysaccharides [1,15,16]. However, this complex process introduces challenges in standardization, as variations in microbial composition [1], storage conditions, and packaging materials [17] significantly impact the final chemical profile and bioactivity.

Despite extensive research on Guang Chenpi’s pharmacological properties, several knowledge gaps remain. First, comprehensive studies systematically evaluating its multi-target bioactivities using advanced in vivo models are limited. Second, the exploration of its combined effects with established bioactive compounds represents an underexplored area. Third, the molecular mechanisms underlying its redox homeostasis and mitochondrial function warrant further investigation.

To address these research gaps, the present study prepared a standardized Guang Chenpi extract (GCE) from three-year-aged materials sourced from Xinhui District, Jiangmen City. The extract was characterized to contain hesperidin (760 mg/L), naringin (240 mg/L), and tangeretin (230 mg/L). We investigated the combination interactions between GCE and two well-established bioactive compounds—ergothioneine (EGT) and polydeoxyribonucleotide (PDRN)—using UVB-irradiated 3D skin tissue models [18]. Furthermore, we employed zebrafish embryo models to comprehensively evaluate GCE’s toxicity profile and multifaceted bioactivities, including antioxidant, wound healing, skin barrier protection, and whitening effects. This study provides new insights into the therapeutic potential of Guang Chenpi in dermatological applications and contributes to the scientific validation of its traditional uses.

2. Results

2.1. Characterization and Quality Control of GCE

The prepared GCE was a brownish liquid with a characteristic odor. Quantitative analysis confirmed the presence of key flavonoids: hesperidin, nobiletin, and tangeretin at concentrations of 242.72 ± 27.29 μg/mL, 127.35 ± 6.11 μg/mL, and 117.20 ± 16.57 μg/mL, respectively (see Supplementary Materials for details). The pH (1% solution, 25 °C) was 4.0–8.0. Microbiological quality was within acceptable limits, with total aerobic microbial count ≤ 100 CFU/g, total combined molds and yeasts ≤ 10 CFU/g, and the absence of Escherichia coli. Safety specifications for heavy metals were also met, with total heavy metals not exceeding 10 mg/kg and arsenic content below 2 mg/kg.

2.2. Combined Effects with EGT and PDRN in UVB-Irradiated Skin Models

2.2.1. Enhanced Depigmentation Activity

GCE demonstrated significant combination effects with EGT and PDRN in reducing melanogenesis (Figure 1). While GCE alone (10 mg/mL) reduced Pmel17 by 23.77%, the combination with EGT (0.2 mg/mL) and PDRN (0.5 mg/mL) enhanced efficacy to 42.71% and 44.27% reductions, respectively (p < 0.01). These results indicate potent enhanced depigmentation activity, surpassing the positive control kojic acid (42.19% reduction).

2.2.2. Enhancement of the Antioxidant Defense System

ROS Scavenging Activity

ROS staining analysis demonstrated that UVB irradiation significantly induced oxidative stress in skin tissues, with the model group exhibiting a substantial increase in ROS fluorescence intensity compared to the normal control (p < 0.001). As illustrated in Figure 2, treatment with GCE (10 mg/mL) alone significantly reduced ROS levels by 46.27% relative to the model control (p < 0.001). The combination therapies exhibited enhanced efficacy: GCE with EGT (10 + 0.2 mg/mL) and GCE with PDRN (10 + 5 mg/mL) reduced ROS levels by 50.31% and 57.76%, respectively (p < 0.001), showing comparable effectiveness to the positive control kojic acid (58.70% reduction). These results confirm the potent ROS scavenging capacity of GCE and its enhanced combination effects with EGT and PDRN.

Increased NNT Level

The antioxidant defense mechanism was further investigated through NNT immunostaining analysis. UVB irradiation significantly decreased NNT concentration by approximately 80% compared to normal skin (p < 0.001). As shown in Figure 3, GCE monotherapy increased NNT levels by 133% relative to the model control. The combination groups showed superior efficacy, with GCE + EGT and GCE + PDRN increasing the NNT signal by 157% and 186%, respectively (p < 0.001). Statistical analysis revealed significant combination interactions between GCE and both EGT and PDRN (p < 0.05), indicating that the combinations potentiate NNT upregulation more effectively than GCE alone.

Upregulation of GSH-PX1 Content

The antioxidant enzyme profile was completed by assessing GSH-PX1 concentration (Figure 4). UVB irradiation caused a significant decrease in GSH-PX1 levels, which was effectively counteracted by all treatment regimens. GCE alone increased GSH-PX1 by 133%, while the combinations with EGT and PDRN further elevated levels by 157% and 186%, respectively (p < 0.001). The GCE + PDRN combination showed a particularly strong combination effect, exceeding the effect of the positive control kojic acid (181% increase).

The coordinated upregulation of key antioxidant components (ROS scavenging, NNT, and GSH-PX1) demonstrates that GCE and its combinations enhance multiple facets of the antioxidant defense system. The significant combination effects observed with EGT and PDRN (p < 0.05) suggest that these combinations act through complementary mechanisms to bolster the endogenous antioxidant capacity. This multi-targeted approach provides a scientific basis for developing advanced formulations against UV-induced oxidative stress and photoaging.

2.3. Acute Toxicity Assessment in Zebrafish Embryos

The developmental toxicity assessment of Guang Chenpi extract (GCE) in zebrafish embryos demonstrated a favorable safety profile. As shown in Figure 5, embryos in the negative control group developed normally without adverse effects, while the positive control (4 mg/mL 3,4-dichloroaniline) induced 35% mortality and 65% malformation. GCE treatment resulted in concentration-dependent toxicity, with 100% mortality observed at 3.03 and 6.67 mg/mL, and 10% mortality at 1.38 mg/mL. Importantly, no significant mortality or developmental abnormalities were detected at concentrations ≤ 0.63 mg/mL, establishing the maximum non-toxic concentration for subsequent assays.

2.4. Multifaceted Bioactivities of GCE in Zebrafish Models

2.4.1. Concentration-Dependent Antioxidant Activity

GCE demonstrated significant reactive oxygen species (ROS) scavenging capacity in a concentration-dependent manner (Figure 6). Compared to the negative control, glutathione (GSH, 0.1 mg/mL) reduced ROS levels by 36.06%. Notably, GCE at concentrations of 0.025, 0.05, 0.1, and 0.15 mg/mL exhibited dose-responsive ROS reductions of 27.57%, 36.43%, 56.11%, and 61.85%, respectively (p < 0.001 vs. control). These results indicate potent antioxidant activity, with higher concentrations showing efficacy superior to the positive control.

2.4.2. Enhanced Wound Healing and Tissue Regeneration

The wound healing assay revealed significant enhancement of tail fin regeneration following GCE treatment (Figure 7). Compared to the model control (219.62 relative length), GCE at 0.005–0.03 mg/mL promoted regeneration by 11.35–27.84% (p < 0.05–0.001). The highest concentration (0.03 mg/mL) showed superior efficacy to the positive control (Rehmannia glutinosa extract, 18.98% enhancement), demonstrating remarkable wound healing properties.

2.4.3. Skin Barrier Protective Effects

GCE significantly protected against lactic acid-induced skin barrier damage in a concentration-dependent manner (Figure 8). Treatment with 0.1–0.6 mg/mL GCE reduced fluorescein sodium penetration by 65.20–89.32% (p < 0.001), comparable to the positive control ectoin (84.75% protection). These results demonstrate potent barrier-enhancing properties relevant to dermatological applications.

2.4.4. Extracellular Matrix Gene Expression Modulation

GCE significantly upregulated extracellular matrix-related gene expression at low concentrations (0.025–0.05 mg/mL), as shown in Figure 9. Collagen genes (col1a1a, col1a1b, and col1a2) were upregulated by 32.33–86.31%, while elastin (elna) expression increased by 19.74–39.44% (p < 0.05–0.001). These findings suggest potent anti-aging and tissue-regenerative properties through extracellular matrix modulation.

2.4.5. Concentration-Dependent Enhancement of Peripheral Blood Circulation

Quantitative assessment of dorsal aorta blood flow velocity, as represented in Figure 10, revealed that the positive control caffeine (0.05 mg/mL) enhanced circulatory function by 13.24% compared to baseline levels (p = 0.0023). GCE administration at concentrations of 0.05, 0.1, and 0.15 mg/mL induced progressive increases in blood flow velocity of 7.62% (p < 0.05), 9.01% (p < 0.05), and 10.20% (p < 0.01), respectively, establishing a clear dose-response relationship.

2.4.6. Dose-Dependent Inhibition of Melanogenesis

As documented in Figure 11, phenylthiourea (PTU, 30 µg/mL) completely suppressed melanogenesis, while kojic acid (2.5 mg/mL) reduced melanin content by 46.53% (p < 0.001). GCE treatment at 0.05, 0.1, and 0.15 mg/mL concentrations induced progressive reductions in melanin synthesis by 8.41%, 9.69%, and 10.85%, respectively (p < 0.001 for all concentrations), establishing a definitive concentration–response relationship.

3. Discussion

Chenpi, as a traditionally revered medicinal and edible substance, has garnered increasing scientific interest due to its rich phytochemical profile and broad bioactivities. Recent research has primarily focused on characterizing its chemical composition, particularly the flavonoid constituents, such as hesperidin, naringin, and tangeretin, which accumulate during the aging process and contribute significantly to its therapeutic properties [19,20,21,22,23,24,25]. The present study aligns with and expands upon this foundation by demonstrating that a standardized Guang Chenpi extract (GCE) exhibits multifaceted dermatological activities, substantiated through rigorous in vivo and in vitro models.

Our investigation reveals that GCE possesses a favorable safety profile alongside potent, dose-dependent bioactivities. The antioxidant efficacy observed—with ROS reduction up to 61.85%—corroborates earlier findings on flavonoid-mediated free radical scavenging [4,24,25]. Notably, this study systematically reports GCE’s capacity to enhance tissue regeneration and fortify the skin barrier, two critical mechanisms in mitigating age-related cutaneous dysfunction. The upregulation of collagen and elastin genes further suggests GCE’s role in extracellular matrix remodeling, addressing key pathways impaired in photoaged skin.

A pivotal finding of this work is the combination interplay between GCE and established bioactive compounds (EGT and PDRN). This combination not only suppressed melanogenesis (via Pmel17 downregulation) but also potently augmented cellular resilience to oxidative stress. The enhanced activation of antioxidant defense systems, particularly through the upregulation of NNT and GSH-PX1, underscores the central role of mitochondrial redox regulation in GCE’s mechanism. As NNT is a key mitochondrial enzyme generating NADPH by utilizing the proton gradient produced by oxidative phosphorylation (OXPHOS), thereby linking redox homeostasis to mitochondrial energy metabolism [26], the involvement of NNT highlights a sophisticated regulatory axis that merits further exploration in the context of redox homeostasis and mitochondrial function.

These findings significantly advance the understanding of traditional natural products in redox biology and cellular protection. The demonstrated efficacy of GCE, particularly through mechanisms involving NNT, underscores its pharmacological potential in targeting oxidative stress and mitochondrial dysfunction. This work bridges traditional knowledge with contemporary mechanistic science, offering a validated, multi-target approach for developing novel interventions against redox-related pathologies.

While this study provides compelling evidence for GCE’s efficacy, certain limitations should be acknowledged. The concentration ranges tested, though pharmacologically relevant, may not fully capture clinical dosing scenarios. Additionally, the exact bioavailability of GCE’s constituents and the precise mechanistic role of NNT in humans remain to be fully characterized. Future work should prioritize elucidating NNT-mediated pathways using genetic or pharmacological approaches, alongside clinical trials to validate these preclinical findings.

4. Materials and Methods

4.1. Chemicals and Reagents

Besides the GCE solution prepared for this study, the following reagents were procured and used: Rehmannia glutinousa Libosch extract (batch number: N103002586) procured from Aladin Group, Shanghai, China; 3,4-dichloroaniline (DCA), caffeine, glutathione, tricaine, ectoin, and lauryl sodium sulfate (SLS) from Sigma-Aldrich, Shanghai, China; H₂DCFDA, M-254 culture medium, newborn calf serum, and GSH-Px1 antibody from Thermo Fisher, Shanghai, China; RNAsimple total RNA kit (Catalog number: DP419) from Tiangen, Beijing, China; SYBR Premix Ex Taq (CN830A) from Takara, Beijing, China; ergothioneine (purity ≥ 95%) from GeneIII; in vitro skin tissue culture medium from Boxi Biology, Guangzhou, China; dimethyl sulfoxide (DMSO) and MTT Cell Proliferation and Cytotoxicity Assay Kit from Biyuntian, Changhai, China; phosphate-buffered saline (PBS) from Solibo, Beijign, China; kojic acid from Yuanye Biotech Biology, Shanghai, China; Pmel17 and NNT antibodies from Santa, Shanghai, China; and paraformaldehyde (PFA) from Biosharp, Beijing, China; PDRN(MW: 5000–15,000 Da, purity ≥ 95%) from ReaLi Tide, Weihai, China.

4.2. GCE Preparation and Quality Control

The Guang Chenpi Extract (GCE) was prepared from authenticated, ≥3-year-old dried pericarps of Citrus reticulata ‘Chachiensis’ (sourced from Jiangmen, China) that were ground into a 100-mesh powder. A 10% (w/v) suspension of the powder in dipropylene glycol was subjected to sequential homogenization steps with additional solvent and purified water. The homogenate was filtered (500-mesh), heated at 100 °C for ≥10 h, and the supernatant was sterile-filtered (0.22 µm) to obtain GCE. The extract was characterized for microbiological quality (total aerobic count, molds/yeasts, E. coli) and heavy metals, and key flavonoids were quantified via LC-MS/MS (Agilent MS QqQ Mass Spectrometer with ESI JetStream, Agilent Technologies, Santa Clara, CA, USA).

4.3. In Vitro Combined Effects Assessment

4.3.1. Skin Tissue Culture and UVB Irradiation

MelaKutis^® 3D skin tissues (24 ± 2 mm²) were cultured in 6-well plates and irradiated with 50 mJ/cm² UVB for 7 consecutive days. Treatment groups received GCE (10 mg/mL), GCE + EGT (10 + 0.2 mg/mL), or GCE + PDRN (10 + 0.5 mg/mL). Given that the stratum corneum barrier enhances topical tolerance [27], these concentrations were set at tenfold the cytotoxic levels determined in prior 2D melanocyte assays (see Supplementary Materials).

4.3.2. ROS Staining

Tissues were stained with 60 µM 1 H₂DCFDA solution for 1 h, fixed using 4% PFA, and cryosections were photographed using an Olympus BX43 fluorescence microscope (Olympus, Tokyo, Japan) and analyzed with Image-Pro^® Plus 6.0 software. The ROS removal rate was calculated as (F_model − F_sample)/F_model) × 100%.

4.3.3. Immunofluorescence Staining

Tissues were fixed in 4% PFA, sectioned, and stained with antibodies against Pmel17, NNT, and GSH-Px1. Imaging was performed using an Olympus BX43 fluorescence microscope and analyzed with Image-Pro^® Plus 6.0 software. The protein increasing rate was calculated as (F_model − F_sample)/F_model) × 100%.

4.4. In Vivo Zebrafish Assays

4.4.1. Zebrafish Maintenance and Ethical Statement

Wild-type AB strain zebrafish (Danio rerio) were maintained under standard conditions at 28.5 °C with a 14/10 h light/dark cycle according to established protocols [28,29]. Embryos were collected by natural spawning and reared in embryo medium. All experimental procedures were approved by the Department of Health, Hong Kong SAR, China (Approval No. 23-86 in DH/HT&A/8/218 Pt.524.10.2023, approved on 24 October 2023).

4.4.2. Acute Toxicity Assessment

Zebrafish embryos at 4–128 cell stage (n = 20 per concentration) were exposed to GCE (0.28–6.67 mg/mL) in 96-well plates until 48 h post fertilization (hpf) [28,29]. Mortality and morphological abnormalities were recorded, with 0.004 mg/mL DCA as positive control.

4.4.3. Antioxidant Activity Evaluation

After 48 hpf, embryos were treated with GCE (0.025–0.15 mg/mL) for 24 h, followed by 10 μM H₂DCFDA staining for 2 h. Fluorescence imaging was performed using Nikon DS-Qi2 microscope (Nikon, Tokyo, Japan) and quantified with ImageJ ij154-win-java8 software. ROS scavenging rate was calculated as: (F_control − F_sample)/F_control × 100%.

4.4.4. Wound Healing Assessment

At 3 dpf, tail fins were amputated surgically, and embryos were treated with GCE (0.005–0.03 mg/mL) for 48 h. Regeneration was quantified using Leica MZ10F stereomicroscope (Leica, Wetzlar, Germany) and ImageJ ij154-win-java8 software. Regeneration rate was calculated as: (L_sample − L_model)/(L_control − L_model) × 100%.

4.4.5. Blood Circulation Analysis

Briefly, 72 hpf embryos were treated with GCE (0.05–0.15 mg/mL) for 2 h, and dorsal aorta blood flow velocity was recorded using Nikon DS-Qi2 microscope and analyzed with EthoVision XT 17 software (Noldus). Enhancing blood circulation rate was calculated as (V_sample − V_control)/V_control) ×100%.

4.4.6. Skin Barrier Protection Test

Embryos at 48 hpf were co-treated with GCE (0.1–0.6 mg/mL) and lactic acid (1.1 mg/mL) for 2 h, then fluorescein sodium (0.5 mg/mL) was added for staining. Barrier integrity was assessed by fluorescence quantification using a fluorescence microscope (Nikon DS-Qi2) imaging and ImageJ analysis. The skin barrier protection rate was calculated as (F_sample − F_model)/F_control − F_model) ×100%.

4.4.7. Gene Expression Analysis

The analysis was performed as previously described [29]. qRT-PCR was performed using the RNAsimple total RNA kit and SYBR Premix Ex Taq. Gene expression of col1a1a, col1a1b, col1a2, and elna was analyzed using the 2^(−ΔΔCt) method with β-actin as a reference gene.

4.4.8. Melanin Inhibition Assay

Embryos (6–8 hpf) were treated with GCE (0.05–0.15 mg/mL) until 48 hpf. Melanin content was quantified by image analysis of transmittance signals as described [29,30].

4.5. Statistical Analysis

Microsoft Excel (version 2410) was used to analyze the data; the results are expressed as the mean ± SD. The comparisons between groups were performed using a two-tailed t-test, and p < 0.05 was considered a significant difference.

5. Conclusions

This study provides comprehensive evidence that Guang Chenpi extract (GCE) is a safe and multi-target natural ingredient with broad pharmacological and dermatological potentials. Through systematic evaluation in zebrafish and 3D tissue models, GCE exhibited low toxicity and concentration-dependent efficacy in enhancing antioxidant defense (ROS reduction up to 61.85%), promoting tissue regeneration (up to 27.84% improvement), strengthening barrier integrity (up to 89.32% protection), and regulating extracellular matrix remodeling. These activities reflect fundamental mechanisms—such as redox homeostasis, cellular repair, and matrix stabilization—that are relevant not only to skin but also to systemic human health. Furthermore, GCE demonstrated notable enhanced protective effects with established bioactive compounds (EGT and PDRN), supporting its role as a potentiator of cellular resilience under stress conditions. These findings underscore the potential of GCE as a multi-functional modulator in preventive health strategies, meriting further investigation into its applications in functional foods, nutraceuticals, and systemic protective formulations. Future studies should focus on elucidating its pharmacokinetics, organ-specific effects, and long-term benefits in models of metabolic, inflammatory, and age-related disorders.