Compositional Analysis of Glycyrrhiza uralensis, G. inflata, and G. glabra after Honey Processing, and the Cardioprotective Effects in Zebrafish Embryos

The “Shang Han Lun” indicates that honey-processed licorice protects the heart better than raw licorice. Ten major constituents in honey-processed licorice samples were quantified. Protective effects of honey-processed licorices against doxorubicin-induced cardiotoxicity were assessed in zebrafish larvae. Network pharmacology analysis based on the ten target constituents was conducted. Results showed glabridin was lowest in honey-processed Gg, while total content of six components (such as liquiritin) was highest in honey-processed Gu, followed by honey-processed Gi, and lowest in honey-processed Gg. Pharmacological results indicated that honey-processed Gu and Gi significantly improved doxorubicin-induced abnormal pericardial edema and increased venous sinus-arterial bulb distance in larvae. The pericardial area was reduced by 23% and 20%, respectively compared to the model group, and the distances reduced to 81% and 83.3% of the model group, respectively. Although improvements in pericardial edema were rare in the honey-processed Gg group, it reversed venous sinus-arterial bulb distance increase. These results indicate that honey-processed Gu and honey-processed Gi can significantly protect zebrafish embryos against the effects of doxorubicin-induced cardiotoxicity, namely, abnormal heart rate, pericardial edema, and elongation of the venous sinus-arterial bulb distance, whereas honey-processed Gg can only significantly reverse the doxorubicin-induced increase in the venous sinus-arterial bulb distance. Network pharmacology analysis predicted that these constituents have potential for the treatment of metabolic abnormalities and cellular senescence related diseases caused by reactive oxygen species induction, linking to Rap1 pathways. Honey-processed Gu and honey-processed Gi had stronger cardioprotective effects on zebrafish embryos than honey-processed Gg possibly because of differences in composition.

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Keywords

licorice from three origins - honey-processed licorice - compositional analysis - cardioprotective effects - Network pharmacology

Abbreviations

DOX: doxorubicin

Gg : Glycyrrhiza glabra

Gi : Glycyrrhiza inflata

GO: Gene Ontology

Gu : Glycyrrhiza uralensis

hpf: hours post-fertilization

HP-Gg : honey-processed Gg

HP-Gi : honey-processed Gi

HP-Gu : honey-processed Gu

HPL : honey-processed licorice

KEGG: Kyoto Encyclopedia of Genes and Genomes

LOQs: limits of quantification

PPI: protein-protein interaction

RSD: the relative standard deviation

SV–BA: the venous sinus-arterial bulb

Introduction

Glycyrrhiza spp. are used worldwide and are mentioned in the pharmacopoeia of China, Russia, and other countries [1]. Licorice the dried root and rhizome of Glycyrrhiza uralensis (Gu), Glycyrrhiza inflata (Gi) or Glycyrrhiza glabra (Gg) is an essential herbal medicine in traditional Chinese medicine. Among them, Gu is widely distributed throughout Inner Mongolia, Gansu, Xinjiang, Qinghai, Shaanxi, Ningxia, Shanxi, and Heilongjiang. Gg is mainly distributed in Xinjiang. In addition, Gi is mainly distributed in Xinjiang and in the northwest region of Gansu [2], [3]. Licorice is a typical representative of a large variety which raw and processed products treat different diseases in clinical practice. During honey processing, the content of flavonoid glycosides, such as liquiritin, tends to increase, whereas glycyrrhizic acid content decreases. The sugar derivative content in honey-processed licorice (HPL) has been shown to be notably higher than that in raw licorice and non-honey-processed licorice [4]. Nevertheless, the specific differences in the composition of licorice from different origins remain unclear and require further comprehensive analysis.

HPL from multiple plant sources have high similarities in chemical composition, therefore, a sensitive and high-throughput animal model suitable for screening is needed to study their differences in arrhythmia efficacy. Zebrafish embryos are transparent which can directly observe the changes of the heart with a microscope. In addition, the cardiovascular system of zebrafish is similar to that of mammals. As a live animal model, zebrafish embryos have been used for high-throughput screening in the development of new drugs for cardiovascular diseases. Through preliminary research, zebrafish embryos as a whole animal are highly suitable for studying the differences of HPL from multiple plant sources in arrhythmic efficacy, and its administration method of water decoction is consistent with clinical utilization. Therefore, this study utilized a doxorubicin (DOX)-induced cardiotoxicity model in zebrafish embryos to explore the enhanced effect of HPL on restoring pulse and calming palpitations.

Numerous studies have revealed the potential of multiple bioactive components in licorice to improve cardiovascular health. For example, liquiritin, one of the primary licorice-derived flavonoids, has been demonstrated to protect against cardiac fibrosis after myocardial infarction and relieve cardiac hypertrophy induced by pressure overload [5], [6]. Li found that licorice-derived liquiritigenin could attenuate isoprenaline-induced myocardial fibrosis in mice through the TGF-β1/Smad2 and AKT/ERK signaling pathways [7]. The different cardiovascular health benefits of licorice, such as Gu, Gi, Gg, and HPL, largely depend on its medicinal ingredients. Nevertheless, research into the cardioprotective effects of various licorice extracts remains limited. Thus, the different efficacies of their pharmaceutical ingredients cannot be scientifically compared. The potential cardiotoxicity risk associated with the common antineoplastic drug DOX has been widely reported and investigated [8]. As alternative therapeutic approaches, natural compounds and traditional Chinese medicine exhibit high efficacy, low cost, and fewer side effects, while also demonstrating promising potential for multi-target effects [9]. Zebrafish have a short developmental time and high genetic similarity to humans, thus, they are frequently utilized as experimental research models [10]. Zebrafish embryos have an intact internal circulatory system and transparent body, which is suitable for studying exogenous toxic effects and the mitigating effects of natural ingredients [11]. Here, we developed a DOX-induced cardiotoxicity model in zebrafish embryos to investigate the cardioprotective effects of licorice.

This study aimed to identify the differences in the medicinal components and cardiac health benefits of HPL from different plant sources. A HPLC technique was employed to quantitatively analyse ten major constituents in licorice from three distinct origins, and the compositional analysis of licorice after honey processing was examined. The licorice extracts were then assessed in the DOX-induced cardiotoxicity model in zebrafish embryos. The associated cardiac parameters were measured to compare the differences in the cardioprotective effects of HPL from three sources, offering theoretical guidance for the clinical application of different types of HPL in the treatment of cardiovascular diseases. HPLC provides modern scientific support for the separation, identification, quantification, and qualitative analysis of herbal medicine components, thus promoting the standardization and precision of herbal medicine research. Network pharmacology, through multidimensional network analysis, reveals the complex molecular networks involved in disease treatment, improving the systematic comprehension of Chinese medicine pharmacological mechanisms [12], [13]. The integration of these two approaches not only offers robust technical support for a deeper understanding of traditional Chinese herbalism pharmacological actions but also lays a solid foundation for the modernization of traditional Chinese medicine.

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Results

The calibration curves, linear ranges, correlation coefficients, and limits of quantification (LOQs) were calculated ([Table 1]). The calibration curves were constructed by plotting the peak area (y) against the concentration (x) of the mixed standard solution, with six duplicates. All ten standards exhibited good linearity of regression coefficients (R ² > 0.999). The LOQs of the analytes ranged from 0.08 385 – 1.767 µg/mL, respectively, indicating high sensitivity. Ten reference solutions at a concentration of 100% were prepared and added to the known sample for recovery testing. The mean recovery and the relative standard deviation (RSD) met the standard requirements. The precision was performed by injecting six replicates of the standard mixtures and the RSD values of the precision experiments were found to not exceed 3.00%. The stability of the sample solution was evaluated by measuring and comparing the peak areas at 0, 2, 4, 8, 12, and 24 hours, and the sample remained stable for 24 hours.

Table 1 The results of chromatographic method validation.
Compound	Regression equation	Linear range (µg/mL)	R ²	LOQs (µg/mL)	Precision	Stability	Repeatability	Recovery
Compound	Regression equation	Linear range (µg/mL)	R ²	LOQs (µg/mL)	(RSD, %, n = 6)	(RSD, %, n = 6)	(RSD, %, n = 6)	%	(RSD, %, n = 6)
Liquiritin apioside	y = 11 769 x − 68 909	26.23 – 1259	0.9998	0.4372	1.28	1.71	2.51	104.5	2.99
Liquiritin	y = 16 493 x − 145 076	42.74 – 1026	1.0000	0.2888	2.33	2.93	1.34	96.21	2.70
Isoliquiritin apioside	y = 24 420 x − 79 994	21.39 – 513.3	1.0000	0.4113	1.26	2.40	2.67	101.3	2.80
Isoliquiritin	y = 30 271 x − 61 449	13.76 – 330.2	1.0000	0.4047	0.93	2.10	2.03	101.2	2.66
Ononin	y = 37 445 x − 17 715	1.758 – 42.19	0.9998	0.2198	1.63	0.72	3.00	99.84	2.93
Licochalcone B	y = 34 166 x − 767.48	1.413 – 33.90	0.9997	0.1765	2.98	2.82	0.67	101.4	2.75
Liquiritigenin	y = 28 571 x − 33 068	5.031 – 120.8	0.9999	0.08385	2.91	1.55	0.38	99.94	2.78
Isoliquiritigenin	y = 61 560 x − 19 825	2.478 – 59.48	0.9999	0.1032	1.95	2.94	2.99	98.70	2.31
Glycyrrhizic acid	y = 9754.3 x − 138 101	72.45 – 1739	0.9999	1.767	0.82	1.16	2.16	100.8	2.84
Glabridin	y = 6743.9 x − 1415.2	1.103 – 26.46	0.9998	1.103	0.70	2.28	2.29	99.02	2.29

The differences in the constituents of HPL from the three origins were compared ([Fig. 1]). The total content of the six components (liquiritin apioside, liquiritin, isoliquiritin apioside, isoliquiritin, liquiritigenin, and isoliquiritigenin) was highest in honey-processed Gu (HP-Gu), followed by honey-processed Gi (HP-Gi), and lowest in honey-processed Gg (HP-Gg). The amount of glabridin in HP-Gi was 2.25 times higher than that in HP-Gg. There was no significant difference in the content of licochalcone B in the three HPLs. Ononin content was relatively lowest in HP-Gi, whereas glabridin content was relatively lowest in HP-Gu.

Fig. 1 Differences in chemical component contents of ten components in HPL from three species (Gu, Gi, and Gg). Data are presented as mean ± SD (n = 3). The vertical coordinates indicate the content of each chemical component per gram of herbs in milligrams. Comparison between each component of licorice from three species, is represented by a, b, and c. Labeling the same character indicates no significant difference, while labeling different characters indicates significant difference (P < 0.05). (HPL, honey-processed licorice; Gu, Glycyrrhiza uralensis.; Gi, G. inflata; Gg, G. glabra.)

HPL samples from different origins were applied to a zebrafish embryonic cardiotoxicity model to reveal their therapeutic effects. Survival analysis curves for acute toxicity tests were obtained ([Fig. 2 a]), revealing the significant acute toxic effect of 8 mg/L DOX on zebrafish embryos in the model group. The survival rate in this group reduced significantly to ~ 50% at 24 hours after exposure and ~ 10% at 72 hours. This effect was ameliorated by the addition of HPL samples from all nine groups. The survival rate in the HPL-treated groups was significantly greater than that of the model group at 24 and 72 hours after exposure. HP-Gu demonstrated the best relief effects. The incubation of zebrafish at the early stages is closely related to the development of the yolk sac and the supplementation of nutrients across the whole body, which is a worthwhile indicator for predicting cardiac development [14]. The hatching rate of the embryos was determined ([Fig. 2 b]). The DOX-induced reduced hatching rate at 48 hours after exposure was significantly ameliorated in the nine HPL-treated groups. The antagonistic effects of HP-Gu and HP-Gi were greater than those of HP-Gg, consistent with the survival analysis results. All zebrafish embryos hatched in all groups at 96 hours after exposure. This outcome indicated that the hatching rate within 48 hours could be inhibited by low DOX concentrations, but the ultimate hatching results were not affected. Thus, HPL can potentially mitigate developmental toxicity, given its protective effects on zebrafish embryo survival and delayed hatching after 8 mg/L DOX exposure. The antagonistic effects of HP-Gu and HP-Gi were greater than those of HP-Gg.

Fig. 2 a Survival curve and b the hatching rates of zebrafish embryos treated with 8 mg/L DOX and/or different origins of HPL. Each experimental group was set up with three biological replicates, with 15 embryos per replicate (n = 15). (HP-Gu, honey-processed Gu.)

Cardiac development parameters were assessed in zebrafish larvae to verify the cardioprotective effect of HPL ([Fig. 3]). First, the heart rate of the zebrafish larvae in each group was recorded and quantified ([Fig. 3 a]). A significant reduction in heart rate occurred in the model group treated with 3.5 mg/L DOX (P < 0.01) compared to that in the control group. This concentration of DOX was used for the cardiac developmental toxicity model as it demonstrated no effect on the survival or hatching of zebrafish in our previous studies. Compared with those in the model group, the heart rates in the HP-Gu and HP-Gi cotreatment groups recovered (P < 0.01), indicating that HP-Gu and HP-Gi have certain mitigating effects. In addition, representative images of zebrafish larvae were captured ([Fig. 3 d]). The larvae in the model group appeared malformed, manifesting as marked pericardial edema, which was tangibly improved in the HPL group, especially in the HP-Gu and HP-Gi groups. The pericardium within the larvae and the venous sinus-arterial bulb (SV–BA) distance were quantified (red dashed line and red solid line, respectively; [Fig. 3 b] and [Fig. 3 c]). Similarly, the DOX-induced abnormal pericardial edema of the larvae was significantly ameliorated by HP-Gu and HP-Gi, and the pericardial area was reduced by 23% and 20%, respectively, compared with that in the model group (P < 0.01). Although improvements in pericardial edema were rare in the HP-Gg group, this treatment significantly reversed the DOX-induced increase in the SV–BA distance (P < 0.05). Similar results were observed in the HP-Gu and HP-Gi groups, where the distances were reduced to 81% and 83.3% of that in the model group, respectively (P < 0.01). These results indicate that HP-Gu and HP-Gi can significantly protected zebrafish embryos against the effects of DOX-induced cardiotoxicity, namely, abnormal heart rate, pericardial edema, and elongation of the SV–BA distance, whereas HP-Gg can only significantly reversed the DOX-induced increase in the SV–BA distance.

Fig. 3 The protective effect of HPLs on cardiac developmental toxicity of zebrafish larvae at 72 hpf induced by 3.5 mg/L DOX. a Heart rate, b Pericardial area, c SV–BA distance, d The representative image of zebrafish in each group. Red dashed lines indicate the pericardial area, and red solid lines indicate the SV–BA distance. (*P < 0.05, **P < 0.01 vs. the zebrafish in control group; ^# P < 0.05, ^## P < 0.01 vs. the zebrafish in DOX group). Each experimental group was set up with three biological replicates, with 15 embryos per replicate (n = 15). (HP-Gu, honey-processed Gu.)

Venn diagram of the 10 major components in licorice targets and disease targets were made. 487 non-duplicated targets of component action and 1771 non-duplicated disease targets were screened through multiple databases, and 157 common targets as the key targets between the drugs and disease were obtained ([Fig. 4 a]). Bioactive ingredient-disease target network diagrams were plotted using Cytoscape 3.6.0 software as shown in [Fig. 4 b]. Licochalcone B, Isoliquiritigenin and Glabridin were more closely related to each target. A protein-protein interaction (PPI) network graph was generated based on intersecting genes, and 48 core target proteins were obtained by secondary filtering based on the network topology parameters ([Fig. 4 c]). To predict the potential pathways of action associated with key components, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed, respectively. The GO analysis yielded a total of 2204 statistically significant entries, of which 1891 were biological processes, 109 were cellular components, and 204 were molecular functions, and the top 10 entries with the smallest P-value for each categorical correction are shown in the figure ([Fig. 4 d]). The KEGG enrichment analysis results demonstrated the top twenty pathways with the smallest corrected P-values ([Fig. 4 e]). Among them, metabolic disease-related AGE-RAGE signaling pathway, the cellular senescence pathway, estrogen signaling pathway and other signaling pathway including Rap1, MAPK, P13K-Akt and reactive oxygen species are thought to be potential pathways by which these components may be used to treat DOX-induced cardiac developmental toxicity in zebrafish embryos. The above results necessitate further validation through fundamental experiments and clinical trials to confirm their reliability.

Fig. 4 Network Pharmacology Analysis: a Venn diagram of targets related to 10 major compounds in licorice and disease-related targets, b Compound-disease target network, c Core PPI network, d KEGG analysis and e GO analysis of the potential targets of 10 major compounds against cardiac developmental toxicity. The size and color are related to the degree value of the target in the network. The larger the size and the darker the color, the higher the degree value of the target. (LQA: liquiritin apioside; LQ: liquiritin; ILQA: isoliquiritin apioside; ILQ: isoliquiritin; ON: ononin; LB: licochalcone B; LG: liquiritigenin; ILG: isoliquiritigenin; GA: glycyrrhizic acid; GL: gabridin.)

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Discussion

Previous studies have revealed that liquiritin, liquiritigenin, and licochalcone from licorice have been shown to benefit heart function [5], [7], [15]. The preliminary experimental results demonstrated that HPL had more beneficial effects on the health of zebrafish heart rate than RL (Fig. 1S, Supporting Information). Our previous study demonstrated that HPL is protective against DOX-induced cardiac dysfunction and pathological damage in zebrafish and that the high distribution of liquiritin, isoliquiritin, and isoliquiritigenin in the heart may be the underlying protective mechanism [11]. In this study, HPL of three origins ameliorated the DOX-induced cardiac developmental toxicity induced in zebrafish larvae, especially HP-Gu and HP-Gi. Specifically, these HPL samples improved the survival and hatchability of zebrafish embryos exposed to high doses of DOX and significantly ameliorated 3.5 mg/L DOX-induced cardiac developmental abnormalities in zebrafish larvae, including pericardial edema, a decreased heart rate, and an increased SV–BA distance. The results of the network pharmacology analysis based on ten target constituents in licorice predicted that these constituents have potential for the treatment of metabolic abnormalities and cellular senescence related diseases caused by reactive oxygen species induction, which are closely related to the Rap1 pathways. Rap1 was reported to inhibit the production of mitochondrial ROS and to reduce susceptibility to early arrhythmia after cardiac depolarization [16]. Oxidative stress and apoptosis are potential factors involved in DOX-induced cardiac injury in zebrafish [17], [18], [19], and HPL may mediate such stress processes in vivo, as both RL and HPL possess potent antioxidant activities [11], [20]. Our findings also supported the results of Wang et al., which indicated that HPL has substantial potential for detoxification [21]. Wang et al. also demonstrated by UPLC-QTOF-MS that liquiritin, isoliquiritin, liquiritin apioside, and glycyrrhizic acid are the most predominant in vivo metabolites [11]. All of these constituents may play a crucial role in the detoxification effects. These findings further reveal the complex interaction between oxidative stress and metabolic pathways in disease development, suggesting that multi-target intervention may be an effective strategy for treating such diseases [22].

As mentioned above, the bioactive ingredients in HPL may have an important contribution to its overall heart-healthy efficacy. In this study, the levels of multiple components in the ten target constituents differed significantly among the three origins of HPL. Generally high levels of glycyrrhizic acid and liquiritin were detected in HPL samples from three different origins. In the previous study, the immunomodulatory activity of HPL was positively correlated with the concentration of glycyrrhizic acid and negatively correlated with acetyl glycyrrhizin (e.g., liquiritin apioside) [23]. Kong et al. revealed a high concentration of glycyrrhetinic acid in mouse serum derived from the metabolic processes of the conversion of glycyrrhizic acid into glycyrrhetinic acid, and more significant pharmacological activities were consequently identified [23]. Studies have also shown that the addition of honey improves heat stability and enhances the bioavailability of pharmaceutical ingredients in licorice [23]. Overall, the synergistic effect of multiple ingredients in HPL greatly contributes to protecting against exogenous cardiac developmental toxicity. Three components (liquiritin apioside, isoliquiritin apioside and liquiritin) are metabolised to isoliquiritin, liquiritigenin, and isoliquiritigenin after absorption, potentially enhancing overall efficacy [24]. Especially, the analysis results showed that the total content of the six components (such as liquiritin) was highest in HP-Gu, followed by HP-Gi, and lowest in HP-Gg. Glabridin in HP-Gi was 2.25 times higher than that in HP-Gg. These may explain why HP-Gg did not significantly ameliorate cardiac developmental abnormalities including reduced heart rate and increased pericardial oedema area in zebrafish larvae.

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Materials and Methods

Reagents and standards

Three plant sources of RL samples and honey were collected by the China Medico Corporation (Tianjin, China) ([Table 2]). They were authenticated by Associate Professor Lihong Chen. Voucher specimens were deposited in the herbarium of the Jiangsu Key Laboratory of Traditional Chinese Medicine Processing.

Table 2 Samples information of licorice from three origins.
No.	Voucher number	Plant sources	Cultivation site
Gu1	H190075001-3	Glycyrrhiza uralensis	Inner mongolia
Gu2	H190080001-3	Glycyrrhiza uralensis	Inner mongolia
Gu3	H190081001-3	Glycyrrhiza uralensis	Inner mongolia
Gg1	J210506-1	Glycyrrhiza glabra	Xinjiang
Gg2	J210506-2	Glycyrrhiza glabra	Xinjiang
Gg3	J210506 – 3	Glycyrrhiza glabra	Xinjiang
Gi1	2 018 120 106	Glycyrrhiza inflata	Gansu
Gi2	2 018 120 107	Glycyrrhiza inflata	Gansu
Gi3	2 018 120 101	Glycyrrhiza inflata	Gansu

Purified water was prepared using a Milli-Q water purification system (Millipore Corporation). Methanol (chromatographic grade) and acetonitrile (chromatographic grade) were purchased from E. Merck. Formic acid (chromatographic grade) and DOX were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Sodium bicarbonate was purchased from Shanghai Hushi Laboratory Equipment Co., Ltd. Methyl cellulose (15 mPa.) was purchased from Macklin Biochemical Technology Co., Ltd. Isoliquiritin apioside, liquiritin apioside, liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, licochalcone B, glabridin, ononin, and glycyrrhizic acid (purity > 98%) were obtained from Chengdu Ruifensi Biotechnology Co., Ltd. The specific chemical structures of the standard reference materials are shown in [Fig. 5]. All other reagents were purchased from commercial sources and were of analytical grade unless otherwise stated.

Fig. 5 Specific chemical structure of ten target analytes.

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Preparation of HPL samples

The HPL samples were prepared using the honey roasting method described in the Chinese Pharmacopoeia (2020 Edition), as reported in previous studies [11]. Specifically, 25 g of refined honey was diluted with a small amount of boiling water. One hundred grams of the RL sample was mixed with the honey water and left to stand for 40 minutes. Subsequently, the mixed sample was quickly stirred for 13.5 minutes using a preheated frying pan, and HPL samples from the corresponding batches were obtained after cooling.

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Component determination of HPL

The dried samples were ground and sifted through a 65-mesh sieve. Portions of HPL powder (0.5 g) were accurately weighed and transferred to conical flasks. Next, 45 mL of 70% methanol (v/v) was added, and the mixture was ultrasonicated for 45 minutes. Methanol was added to a final volume of 50 mL to obtain the methanol extract. Finally, the extract solution was centrifuged at 6, 200 ×g for 10 minutes, and the supernatant was collected and filtered through a 0.2 µm membrane before HPLC analysis.

Ten reference standards, namely, liquiritin apioside, liquiritin, isoliquiritin apioside, isoliquiritin, ononin, licochalcone B, liquiritigenin, isoliquiritigenin, glycyrrhizic acid, gabridin, were weighed and dissolved in methanol to obtain a stock reference solution. The stock solution was diluted with methanol to create a series of working reference solutions with varying concentrations. All stock and working reference solutions were stored at 4 °C until analysis.

Quantitative analysis was performed using a HPLC system consisting of an autosampler, a quaternary pump, a column compartment, and a diode array detector. Chromatographic separation was conducted using a Waters Cortecs C18 column (4.6 mm × 150 mm, 2.7 µm). The type of chromatographic mobile phase, column temperature, and detection wavelength were modified to obtain the optimal chromatographic conditions. The optimal mobile phase was acetonitrile (A) – 0.2% formic acid (B) (50 : 50, v/v) at a flow rate of 0.56 mL · min⁻¹. The gradient elution procedure was as follows: 0 – 5 minutes, 90 – 85% B; 5 – 5.2 minutes, 85 – 80% B; 5.2 – 20 minutes, 80 – 74% B; 20 – 30 minutes, 74 – 70% B; and 30 – 65 minutes, 70 – 30% B. The column temperature was maintained at 35 °C, and the injection volume was 5 µL. Fig. 2S (Supporting Information) shows the typical separation of the mixed standard (a) and HPL (b) obtained at 250 nm for ononin and glycyrrhizic acid; 276 nm for liquiritin apioside, liquiritin and liquiritigenin; 300 nm for glabridin;and 376 nm for isoliquiritin apioside, isoliquiritin, licochalcone B and isoliquiritigenin at the optimized chromatographic conditions.

The proposed HPLC analysis method was validated according to the requirements specified in the Chinese Pharmacopoeia (2020 edition) using calibration curves, LOQs, precision, stability, repeatability, and recovery of all reference compounds. The recovery was calculated using the following formula:

Recovery\ (%)\ =100\times {detected\ amount\hbox{‐}original\ amount\over{spiked\ amount}}

The content in the HPL was calculated after subtracting the weight of honey added (25% honey, in accordance with the 2020 edition of the Chinese Pharmacopoeia).

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Pharmacodynamic experiment in zebrafish embryos

The wild-type AB zebrafish lines utilized in this study were provided by the Nanjing Institute of Environmental Science. The zebrafish were maintained in an automatic housing system (ESEN) under controlled conditions of 27 ± 0.5 °C and a 14/10-hour light/dark cycle. Adult zebrafish were fed live brine shrimp twice daily. Male and female zebrafish were placed in a breeding tank at a 1 : 1 ratio the night before fertilization and separated by a divider. Mating was induced in the morning by turning on the light, and the eggs were collected within 2 hours. The embryos were rinsed with fresh water three times and transferred to fresh water tanks at 27 °C for subsequent experiments.

HPL slices from different origins were added in ten times the volume of water and heated to boiling. The solution was boiled for 30 minutes and then filtered. The filter residue was collected, eight times the volume of water was added, and the residue was treated as described above. Two batches of filtrate were combined and centrifuged at 9, 600 ×g for 10 minutes to obtain the HPL solutions.

Exposure experiments were conducted by exposing zebrafish embryos to different concentrations of sample solutions at 6 hours post-fertilization (hpf). DOX was chosen as the positive control for constructing the zebrafish embryonic cardiotoxicity model. Embryos were placed in 6-well plates at a density of 15 embryos per well, with three replicates per group, and subjected to different treatments to investigate the effects of HPL solutions on heart development [25], [26], [27]. The control group was exposed to aquaculture water, the model group to 8 mg/L DOX, and three experimental groups to a combination of DOX and HP-Gu, HP-Gi, HP-Gg. All groups were incubated at 28 °C in a light incubator with controlled light and temperature. The survival and hatching status of the zebrafish embryos were monitored and analyzed after intervention for 24, 48, 72, and 96 hours, and the effect of HPL solutions on DOX-induced zebrafish embryo death was also determined.

Zebrafish embryos at 48 hpf were used for exposure experiments. The control group was treated with aquaculture water, while the model group was treated with 3.5 mg/L DOX. The experimental groups received 27 mg/L of HP-Gu, HP-Gi, or HP-Gg and DOX at a concentration of 3.5 mg/L. The selected concentration of HPL showed no adverse effect on zebrafish embryos in the preliminary studies. After 24-hour intervention, the heartbeat rates of the zebrafish larvae were recorded over 1 minute using a body microscope (SMZ25, Nikon). Ten larval tails were counted, and the recorded videos were manually counted three times. Simultaneously, images of the zebrafish larvae were captured. The distances from SV–BA and the pericardial area were quantified using the Image J software to evaluate the cardiac development of zebrafish larvae in each group. Three biological replicates were established per experimental group, with 15 embryos per replicate.

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Target network analysis

Target prediction of ten compounds was performed through TCMSP, PharmMapper, and Swiss Target Prediction databases, and the top-ranked target information was screened to create a bioactive ingredient target dataset. DrugBank (https://go.drugbank.com/), OMIM (https://omim.org/), GeneCards (https://www.genecards.org/), and PharmGKB (https://www.pharmgkb) databases were combined to analyse the target datasets. The databases were searched with the keyword “Adriamycin-induced cardiotoxicity” and downloaded, merged and de-duplicated to create a disease-related target set. The target of the bioactive ingredient of licorice and the disease-related target were input in the Weishengxin website (http://www.bioinformatics.com.cn/) to draw a Veen diagram and mapped to the possible targets of the compounds for the disease treatment.

The intersected targets were imported into a STRING (https://www.string-db.org/) to obtain the PPI network of intersecting targets. The active drug ingredients and the intersection targets were introduced into Cytoscape 3.6.0 to build a visual network, and the Centiscape 2.2 plugin in the software was used to screen core target proteins by analyzing the network topological parameters such as betweenness, closeness and degree. Key targets were imported into the Metascape website for GO and KEGG pathway enrichment analysis, respectively [28]. The GO enrichment was visualized by selecting the top 10 entries with the smallest P value and KEGG enrichment results were visualized by selecting the top 20 entries with the smallest P value. The key nodes and signaling pathways involved were explored.

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Statistical analysis

The experiments were conducted in triplicate, and the data were presented as the mean ± standard deviation (Χ̅ ± SD). Statistical analyses were performed using GraphPad Prism 9.5. Differences between two groups were determined using a Studentʼs t-test, while those among three or more groups were analyzed using a one-way analysis of variance (ANOVA). P values < 0.05 (*) and P < 0.01 (**) were considered to indicate statistical significance.

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Contributorsʼ Statement

Data collection: Wenxin Wang; design of the work: Wenxin Wang, Lihong Chen; statistical analysis: Jiayi Wang, Binghan Liu; analysis and interpretation of the data: Jiayi Wang, Wenxin Wang, Guangchao Yang, Xiaoyu Fan; drafting the manuscript: Jiayi Wang, Guangchao Yang, Wenxin Wang, Binghan Liu, Xiaoyu Fan, Shucen Liu; critical revision of the manuscript: Jiayi Wang, Tulin Lu, Lihong Chen, Jining Liu.

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Conflict of Interest

The authors declare that they have no conflict of interest.

Supporting Information

Supporting Information includes additional experimental data: Fig. 1S – The therapeutic effects of RL and HPL on beats per minute in zebrafish embryos; Fig. 2S – Typical chromatograms of mixed standard and HPL.

Supporting Information

References
1 Jiang L, Akram W, Luo B, Hu S, Faruque MO, Ahmad S, Yasin NA, Khan WU, Ahmad A, Shikov AN, Chen J, Hu X. Metabolomic and pharmacologic insights of aerial and underground parts of Glycyrrhiza uralensis Fisch. ex DC. for maximum utilization of medicinal resources. Front Pharmacol 2021; 12: 658670

PubMed Search in Google Scholar
2 Jiang M, Zhao S, Yang S, Lin X, He X, Wei X, Song Q, Li R, Fu C, Zhang J, Zhang Z. An “essential herbal medicine”–Licorice: A review of phytochemicals and its effects in combination preparations. J Ethnopharmacol 2020; 249: 112439

PubMed Search in Google Scholar
3 Ding Y, Brand E, Wang W, Zhao Z. Licorice: Resources, applications in ancient and modern times. J Ethnopharmacol 2022; 298: 115594

PubMed Search in Google Scholar
4 Ota M, Xu F, Li YL, Shang MY, Makino T, Cai SQ. Comparison of chemical constituents among licorice, roasted licorice, and roasted licorice with honey. J Nat Med 2018; 72: 80-95

PubMed Search in Google Scholar
5 Aiyasiding X, Liao HH, Feng H, Zhang N, Lin Z, Ding W, Yan H, Zhou ZY, Tang QZ. Liquiritin attenuates pathological cardiac hypertrophy by activating the PKA/LKB1/AMPK pathway. Front Pharmacol 2022; 13: 870699

PubMed Search in Google Scholar
6 Han X, Yang Y, Zhang M, Li L, Xue Y, Jia Q, Wang X, Guan S. Liquiritin protects against cardiac fibrosis after myocardial infarction by inhibiting CCL5 expression and the NF-kappaB signaling pathway. Drug Des Devel Ther 2022; 16: 4111-4125

PubMed Search in Google Scholar
7 Li L, Fang H, Yu YH, Liu SX, Yang ZQ. Liquiritigenin attenuates isoprenaline-induced myocardial fibrosis in mice through the TGF-β1/Smad2 and AKT/ERK signaling pathways. Mol Med Rep 2021; 24: 686

PubMed Search in Google Scholar
8 Heidari S, Mehri S, Hosseinzadeh H. The genus Glycyrrhiza (Fabaceae family) and its active constituents as protective agents against natural or chemical toxicities. Phytother Res 2021; 35: 6552-6571

PubMed Search in Google Scholar
9 Wang W, Li H, Shi Y, Zhou J, Khan GJ, Zhu J, Liu F, Duan H, Li L, Zhai K. Targeted intervention of natural medicinal active ingredients and traditional Chinese medicine on epigenetic modification: Possible strategies for prevention and treatment of atherosclerosis. Phytomedicine 2024; 122: 155139

PubMed Search in Google Scholar
10 Song Z, Zhang Y, Zhang H, Rajendran RS, Wang R, Hsiao CD, Li J, Xia Q, Liu K. Isoliquiritigenin triggers developmental toxicity and oxidative stress-mediated apoptosis in zebrafish embryos/larvae via Nrf2-HO1/JNK-ERK/mitochondrion pathway. Chemosphere 2020; 246: 125727

PubMed Search in Google Scholar
11 Wang W, Yu Y, Chen H, Sun P, Lu L, Yan S, Liu X, Lu T, Li W, Liu J, Chen L. Anti-arrhythmia potential of honey-processed licorice in zebrafish model: Antioxidant, histopathological and tissue distribution. J Ethnopharmacol 2023; 316: 116724

PubMed Search in Google Scholar
12 Duan H, Wang W, Shi Y, Wang L, Khan GJ, Luo M, Zhou J, Yang J, He C, Li F, Hu H, Zhai K. Anti-colorectal cancer actions of Glycyrrhiza uralensis Fisch. and its underlying mechanism via HPLC integration and network pharmacological approaches. Phytomedicine 2025; 138: 156370

PubMed Search in Google Scholar
13 Duan H, Li H, Liu T, Chen Y, Luo M, Shi Y, Zhou J, Rashed MMA, Zhai K, Li L, Wei Z. Exploring the molecular mechanism of Schisandrin C for the treatment of atherosclerosis via the PI3K/AKT/mTOR autophagy pathway. ACS Omega 2024; 9: 32920-32930

PubMed Search in Google Scholar
14 Yang G, Yang Q, Beta T, Liu Q, Zhu Z, Shen F. Protective effects of melanoidins from black garlic on zearalenone-induced toxicity in zebrafish embryonic developmental model. Comp Biochem Physiol C Toxicol Pharmacol 2024; 276: 109789

PubMed Search in Google Scholar
15 Lin JH, Yang KT, Ting PC, Lee WS, Lin DJ, Chang JC. Licochalcone a improves cardiac functions after ischemia-reperfusion via reduction of ferroptosis in rats. Eur J Pharmacol 2023; 957: 176031

PubMed Search in Google Scholar
16 Yang Z, Kirton HM, Al-Owais M, Thireau J, Richard S, Peers C, Steele DS. Epac2-Rap1 signaling regulates reactive oxygen species production and susceptibility to cardiac arrhythmias. Antioxid Redox Signal 2017; 27: 117-132

PubMed Search in Google Scholar
17 Kitakata H, Endo J, Ikura H, Moriyama H, Shirakawa K, Katsumata Y, Sano M. Therapeutic targets for DOX-induced cardiomyopathy: Role of apoptosis vs. ferroptosis. Int J Mol Sci 2022; 23: 1414

PubMed Search in Google Scholar
18 Rawat PS, Jaiswal A, Khurana A, Bhatti JS, Navik U. Doxorubicin-induced cardiotoxicity: An update on the molecular mechanism and novel therapeutic strategies for effective management. Biomed Pharmacother 2021; 139: 111708

PubMed Search in Google Scholar
19 Zhang X, Hu C, Kong CY, Song P, Wu HM, Xu SC, Yuan YP, Deng W, Ma ZG, Tang QZ. FNDC5 alleviates oxidative stress and cardiomyocyte apoptosis in doxorubicin-induced cardiotoxicity via activating AKT. Cell Death Differ 2020; 27: 540-555

PubMed Search in Google Scholar
20 Zhou Q, Zhang S, Geng X, Jiang H, Dai Y, Wang P, Hua M, Gao Q, Lang S, Hou L, Shi D, Zhou M. Antioxidant effects of roasted licorice in a zebrafish model and its mechanisms. Molecules 2022; 27: 7743

PubMed Search in Google Scholar
21 Wang Y, Ning Y, He T, Chen Y, Han W, Yang Y, Zhang CX. Explore the potential ingredients for detoxification of honey-fired licorice (ZGC) Based on the metabolic profile by UPLC-Q-TOF-MS. Front Chem 2022; 10: 924685

PubMed Search in Google Scholar
22 Zhai K, Deng L, Wu Y, Li H, Zhou J, Shi Y, Jia J, Wang W, Nian S, Jilany Khan G, El-Seedi HR, Duan H, Li L, Wei Z. Extracellular vesicle-derived miR-146a as a novel crosstalk mechanism for high-fat induced atherosclerosis by targeting SMAD4. J Adv Res 2024; [Epub ahead of Print] + doi:10.1016/j.jare.2024.08.012

Crossref PubMed Search in Google Scholar
23 Kong S, Li P, Verpoorte R, Li M, Dai Y. Chemical and pharmacological difference between honey-fried licorice and fried licorice. J Ethnopharmacol 2023; 302: 115841

PubMed Search in Google Scholar
24 Sun P, Chen H, Fan X, Wang J, Lu L, Yang G, Liu J, Yao W, Ding F, Ding J, Liu J, Lu T, Chen L. Exploring the effective components of honey-processed licorice (Glycyrrhiza uralensis Fisch.) in attenuating Doxorubicin-induced myocardial cytotoxicity by combining network pharmacology and in vitro experiments. J Ethnopharmacol 2024; 329: 118178

PubMed Search in Google Scholar
25 Jeon HJ, Kim C, Kim K, Lee SE. Piperlongumine treatment impacts heart and liver development and causes developmental delay in zebrafish (Danio rerio) embryos. Ecotoxicol Environ Saf 2023; 258: 114995

PubMed Search in Google Scholar
26 Cheng Y, Wu X, Nie X, Wu Y, Zhang C, Lee SMY, Lv K, Leung GPH, Fu C, Zhang J, Li J. Natural compound glycyrrhetinic acid protects against doxorubicin-induced cardiotoxicity by activating the Nrf2/HO-1 signaling pathway. Phytomedicine 2022; 106: 154407

PubMed Search in Google Scholar
27 Sun X, Sun P, Zhen D, Xu X, Yang L, Fu D, Wei C, Niu X, Tian J, Li H. Melatonin alleviates doxorubicin-induced mitochondrial oxidative damage and ferroptosis in cardiomyocytes by regulating YAP expression. Toxicol Appl Pharmacol 2022; 437: 115902

PubMed Search in Google Scholar
28 Tan H, Chen J, Li Y, Li Y, Zhong Y, Li G, Liu L, Li Y. Glabridin, a bioactive component of licorice, ameliorates diabetic nephropathy by regulating ferroptosis and the VEGF/Akt/ERK pathways. Mol Med 2022; 28: 58

Crossref PubMed Search in Google Scholar

Correspondence

Prof. Tulin Lu

School of Pharmacy

Nanjing University of Chinese Medicine

138 Xianlin Avenue

210023 Nanjing (Jiangsu)

China

Phone: + 86 (25) 85811072

Email: ltl2021@njucm.edu.cn

A. P. Lihong Chen