Exocarpium Citri Grandis ameliorates LPS-induced acute lung injury by suppressing inflammation, NLRP3 inflammasome, and ferroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Exocarpium Citri Grandis (ECG), the epicarp of C. grandis 'Tomentosa' which is also known as Hua-Ju-Hong in China, has been widely used for thousands of years to treat inflammatory lung disorders such as asthma, and cough as well as dispelling phlegm. However, its underlying pharmacological mechanisms in acute lung injury (ALI) remain unclear. AIM OF THE STUDY: To explore the therapeutic effect of ECG on ALI and reveal the potential mechanisms based on experimental techniques in vivo and in vitro. MATERIALS AND METHODS: Lipopolysaccharides (LPS) induced ALI in mice and induced RAW 264.7 cell inflammatory model were established to investigate the pharmacodynamics of ECG. ELISA kits, commercial kits, Western Blot, qPCR, Hematoxylin and Eosin (H&E) staining, immunohistochemistry, and immunofluorescence technologies were used to evaluate the pharmacological mechanisms of ECG in ameliorating ALI. RESULTS: ECG significantly attenuated pulmonary edema in LPS-stimulated mice and decreased the levels of IL1ß, IL6, and TNF-α in serum and BALF, reduced MDA and iron concentration as well as increased SOD and GSH levels in lung tissues, and also decreased the ROS level in BALF and Lung tissue. Further pharmacological mechanism studies showed that ECG significantly inhibited mRNA expression of inflammatory signaling factors and chemokines, and down-regulated the expression of TLR4, MyD88, NF-κB p65, NF-κB p-p65 (S536), COX2, iNOS, Txnip, NLRP3, ASC, Caspase-1, JAK1, p-JAK1 (Y1022), JAK2, STAT1, p-STAT1 (S727), STAT3, p-STAT3 (Y705), STAT4, p-STAT4 (Y693), and Keap1, and also up-regulated the expression of Trx-1, Nrf2, HO-1, NQO1, GPX4, PCBP1, and SLC40A1. In the LPS-induced RAW264.7 cell inflammatory model, ECG showed similar results to animal experiments. CONCLUSIONS: Our results showed that ECG alleviated ALI by inhibiting TLR4/MyD88/NF-κB p65 and JAK/STAT signaling pathway-mediated inflammatory response, Txnip/NLRP3 signaling pathway-mediated inflammasome activation, and regulating Nrf2/GPX4 axis-mediated ferroptosis. Our findings provide an experimental basis for the application of ECG.

Selection and evaluation of quality markers for the regulation of PXR-CYP3A4/FXR-LXRα by Exocarpium Citri Grandis for the treatment of hyperlipidaemia with dispelling blood stasis and removing phlegm.

Hyperlipidaemia is described as "excessive phlegm" and "blood stasis" in the classic theory of traditional Chinese medicine. Exocarpium Citri Grandis has the effect of dispelling blood stasis and removing phlegm, which can better meet the treatment needs of this disease. However, there is still a lack of focus and depth in the study of the chemical composition of this medicine, and the correlation between the study of relevant medicinal substances and the efficacy of dispelling stasis and removing phlegm is insufficient. To address this issue, this study was carried out to validate the overall efficacy and identify and determine the chemical composition of Exocarpium Citri Grandis. The regulatory mechanism of the PXR-CYP3A4/FXR-LXRα pathway and its active ingredients were screened, and a pharmacokinetic study of active ingredients was performed. The obtained multidimensional data were statistically analysed and comprehensively evaluated. The quality marker of Exocarpium Citri Grandis in the treatment of hyperlipidaemia based on the PXR-CYP3A4/FXR-LXRα mechanism to exert the efficacy of dispelling blood stasis and removing phlegm was finally determined. Based on the above experiments, we identified 27 compounds from the ethanol extract of Exocarpium Citri Grandis. Among them, naringenin, meranzin hydrate, apigenin, caffeic acid phenethyl ester, anacardiin, hesperidin and naringin can significantly regulate all or part of the targets in the PXR-CYP3A4/FXR-LXRα pathway. It also has suitable content and pharmacokinetic characteristics in vivo. In conclusion, this study established quality markers to characterize the efficacy of Exocarpium Citri Grandis in dispelling blood stasis and removing phlegm, which provides a scientific basis for the targeted evaluation of the hypolipidaemic activity of this medicinal plant.

Exocarpium Citri Grandis alleviates the aggravation of NAFLD by mitigating lipid accumulation and iron metabolism disorders.

ETHNOPHARMACOLOGICAL RELEVANCE: Exocarpium Citri grandis (ECG, Huajuhong in Chinese), the epicarp of C. grandis 'Tomentosa', has been used for hundreds of years as an anti-inflammatory, expectorant, hypoglycemic, and lipid-lowering medication in China. Nevertheless, there have been few papers that have explored the mechanism behind ECG's hypolipidemic characteristics from the perspective of treating nonalcoholic fatty liver disease (NAFLD). AIM OF STUDY: The purpose of our study was to confirm the therapeutic and preventative effects of ECG in NAFLD by regulating lipid accumulation and iron metabolism, and to explore the specific mechanism of ECG in enhancing hepatic iron transport and excretion capabilities. STUDY DESIGN: We constructed a NAFLD model by feeding male C57BL/6 J mice with a high-fat diet for 12 weeks. Mice were gavaged with ECG beginning in the seventh week of modeling, and three dosage gradients were established: low dose group (2.5 g/kg/d), medium dose group (5 g/kg/d) y, and high dose group (10 g/kg/d) until the end of model construction in week 12. MATERIALS AND METHODS: We used network pharmacology to analyze the relationship between ECG and NAFLD. In addition, we constructed a nonalcoholic fatty liver disease model by feeding male C57BL/6 J mice a high-fat diet for 12 weeks. Finally, lipid accumulation, iron accumulation, inflammation and oxidative stress were evaluated by serological index detection, histological detection, immunofluorescent and immunohistochemical staining, and western blotting. RESULTS: Network pharmacology confirmed the treatment effect of ECG in NAFLD. Three active components of ECG, including Naringenin, Naringin and Neohesperidin, were detected by UHPLC-HRMS analysis. The results of serum TC, TG, LDL concentration, HE staining, Oil red staining and Nile red staining demonstrated that ECG could improve lipid metabolism disorders. The results of serum iron concentration, liver tissue iron concentration, iron metabolism-related proteins Ferritin light chain, Ferroportin1, Transferrin receptor, and Transferrin demonstrated that ECG improved the iron transport and storage capacities of hepatic cells. CONCLUSIONS: Our results demonstrated that ECG relieved liver injury by inhibiting lipid accumulation and iron accumulation in NAFLD.

Optimization of extraction flavonoids from Exocarpium Citri Grandis and evaluation its hypoglycemic and hypolipidemic activities.

ETHNOPHARMACOLOGICAL RELEVANCE: Exocarpium Citri Grandis (ECG; Huajuhong in Chinese) is a precious traditional Chinese medicine with a history of hundreds of years in China. It has been demonstrated to possess numerous pharmacological properties, including antitussive, expectorant, anti-inflammatory, hypoglycemic, and hypolipidemic. However, no in-depth report exists on the hypoglycemic and hypolipidemic properties of ECG. AIM OF THE STUDY: This study aimed to evaluate the hypoglycemic and hypolipidemic properties of ECG flavonoids extract in vitro and in vivo so as to lay the foundation for further researches in this field. MATERIALS AND METHODS: Total flavonoids (TF) and naringin were separately extracted from ECG, and the components of TF were identified by HPLC-MS. The antioxidant capacities of TF and naringin were determined by 2,2,1-diphenyl-1-picrylhydrazyl (DPPH)-free radical scavenging tests, and digestive enzymes activity inhibition assays in vitro in order to evaluate their hypoglycemic properties. Furthermore, diabetic mice experiments were performed to assess the hypoglycemic and hypolipidemic properties of TF and naringin in vivo. RESULTS: Five compounds were identified from TF, including naringin, rhoifolin, poncirin, bergaptol, and naringenin. The half maximal inhibitory concentration (IC50) of TF and naringin to DPPH-free radicals were 0.269 and 1.946 mg/mL, respectively. TF and naringin demonstrated a certain inhibitory effect on α-glucosidase and a weaker inhibitory effect on α-amylase. The results of animal experiments showed that TF and naringin had no significant effect on the blood glucose levels, but they could lead to significant (p < 0.05 or p < 0.01) increase in the serum insulin level and high-density lipoprotein cholesterol (HDL-C) levels with concomitant reduction in the total cholesterol (TC), total triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) levels in diabetic mice. In addition, TF and naringin could reduce the liver index of diabetic mice (p < 0.01) and reduce the kidney index at low doses (p < 0.05). CONCLUSIONS: Our study revealed that the hypoglycemic and hypolipidemic activities of TF is mainly derived from naringin, and other active ingredients in TF also have the effects of alleviating oxidative stress, inhibiting digestive enzyme activity and reducing blood lipids. Our results thus provide a scientific basis for the application of ECG in antidiabetic treatment.

Effects of Total Flavonoids from Exocarpium Citri Grandis on Air Pollution Particle-Induced Pulmonary Inflammation and Oxidative Stress in Mice.

Exocarpium Citri Grandis (ECG) is a famous traditional Chinese medicine, which has been commonly used to alleviate cough and phlegm for more than several hundred years, and total flavonoids are the main effective components of this medicine. This study investigated the effects of total flavonoids from ECG (TFECG) on pulmonary inflammation and oxidative stress induced by PM2.5 in mice. Model mice received an intratracheal instillation of PM2.5 (10 mg/mL) once at day 0. Bronchoalveolar lavage fluid (BALF) was collected after 72 hr to measure the total number of white blood cell (WBC), neutrophils (NEUT), lymphocytes (LYMPH), and monocytes (MONO). The levels of tumor necrosis factor-α (TNF-α), interleukin 1ß (IL-1ß), interleukin-6 (IL-6), and interleukin-18 (IL-18) in BALF were quantified by using enzyme-linked immunosorbent assay kits. Lung tissues were used to determine the contents of total protein (TP), malondialdehyde (MDA), nitric oxide (NO), reduced glutathione (GSH), oxidized glutathione (GSSG) and the activities of superoxide dismutase (SOD), lactate dehydrogenase (LDH), Glutathione Peroxidase (GSH-Px), and inducible nitric oxide synthase (iNOS). We found that TFECG significantly inhibited PM2.5 -stimulated overproduction of TNF-α, IL-1ß, IL-6, and IL-18 and increased the numbers of WBC, NEUT, LYMPH, and MONO in BALF. TFECG observably relieved the PM2.5 -induced increases in the contents of TP, MDA, and NO, and the activities of LDH and iNOS. TFECG also alleviated PM2.5 -induced decreases in the activities of iNOS and GSH-Px as well as GSH/GSSG ratio. The results indicate that TFECG has anti-inflammatory and antioxidant activities, which may potentially contribute to the treatment of PM2.5 -induced lung injury. PRACTICAL APPLICATION: Exocarpium Citri Grandis (ECG) is rich in flavonoids, which are beneficial to improve anti-inflammation and antioxidant capacity. We proved that total flavonoids of ECG had a positive therapeutic effect on PM2.5 -induced lung injury, which expands the potential applications of ECG in the dietary supplement industries.

UFLC-Q-TOF-MS/MS-Based Screening and Identification of Flavonoids and Derived Metabolites in Human Urine after Oral Administration of Exocarpium Citri Grandis Extract.

Exocarpium Citri grandis (ECG) is an important Traditional Chinese Medicine (TCM) for the treatment of cough and phlegm, and the flavonoids contained were considered the main effective components. To date, the systematic chemical profiling of these flavonoids and derived in vivo metabolites in human have not been well investigated. ECG was extracted using boiling water and then provided to volunteers for oral administration. Following the ingestion, urine samples were collected from volunteers over 48 h. The extract and urine samples were analyzed using ultra-fast liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry (UFLC-Q-TOF-MS/MS) system to screen and identify flavonoids and derived in vivo metabolites. A total of 18 flavonoids were identified in the ECG extract, and 20 metabolites, mainly glucuronide and sulfate conjugates, were screened in urine samples collected post consumption. The overall excretion of naringenin metabolites corresponded to 5.45% of intake and occurred mainly within 4-12 h after the ingestion. Meanwhile, another 29 phenolic catabolites were detected in urine. Obtained data revealed that flavonoids were abundant in the ECG extract, and these components underwent extensive phase II metabolism in humans. These results provided valuable information for further study of the pharmacology and mechanism of action of ECG.

Antitussive, expectorant and anti-inflammatory activities of different extracts from Exocarpium Citri grandis.

ETHNOPHARMACOLOGICAL RELEVANCE: Exocarpium Citri grandis (C. grandis, Huajuhong in Chinese), the epicarp of C. grandis 'Tomentosa', is used as an antitussive, expectorant and anti-inflammatory drug for hundreds of years in China. The study was aimed at evaluating the antitussive, expectorant and anti-inflammatory effects of different extracts of C. grandis, providing experimental evidence for its traditional use, and laying a foundation for its further researches. MATERIALS AND METHODS: Crude drugs of C. grandis were extracted with four kinds of solvents (water, 50% ethanol, 70% ethanol and 90% ethanol) in reflux conditions, respectively. Solutions were concentrated in reduced pressure and lyophilized in vacuum to yield the aqueous extract, 50% ethanolic extract, 70% ethanolic extract, and 90% ethanolic extract of C. grandis. Antitussive evaluations were carried out with ammonia liquor induced mice cough; expectorant effects were tested with phenol red secretion experiments in mice; anti-inflammatory effects were assessed by murine model of xylene induced ear edema in mice. RESULTS: Only aqueous and 70% ethanolic extracts of C. grandis displayed significant antitussive, expectorant and anti-inflammatory activities. Aqueous extract of C. grandis significantly decreased cough frequency caused by ammonia liquor, increased phenol red secretion and inhibited the development of ear edema in anti-inflammatory assay at the dose of 1005 mg/kg (P<0.05). However, aqueous extract of C. grandis did not lengthened the cough period. It was worth noting that, 70% ethanolic extract of C. grandis showed strong effect of decreasing cough frequency, prolonging cough period, increasing phenol red secretion and decreasing the extent of ear edema at the dose of 493 mg/kg (P<0.001). The low, middle, and high dose (247, 493, and 986 mg/kg) of 70% ethanolic extract of C. grandis showed significant antitussive, expectorant and anti-inflammatory effects in good dose dependant manner. CONCLUSION: The results supported the folk use of C. grandis (decoction of C. grandis) with scientific evidence, and indicated that the 70% ethanolic extract of C. grandis might have better effects of antitussive, expectorant and anti-inflammatory than those of aqueous extract. Further investigation needs to be conducted to study the bioactive constituents of 70% ethanolic extract of C. grandis and the mechanism of observed antitussive, expectorant and anti-inflammatory activities.

Characteristics of Naringenin Contents in Exocarpium Citri Grandis / 广州中医药大学学报

【Objective】 To assay the naringenin content in different medicinal materials of Exocarpium Citri Grandis (ECG). 【Methods】 High performance liquid chromatography (HPLC) was applied to determine the naringenin content in different medicinal materials of ECG. The chromatographic conditions were: gradient elution being performed with methanol as mobile phase A and water - acetic acid (61: 4) as mobile phase B, flowing rate being 1.0 mL/min; detecting wavelength at 283 nm. 【Results】 In the rage of 0.088～0.880 ?g, naringenin showed a good linearity with the chromatographic peak area, r=0.9999, average recovery rate was 95.49% and RSD=2.00% (n=5). The naringenin content in 23 different samples of ECG was in the range of 0.1～1.0 mg?g~(-1). 【Conclusion】 It is the first time to establish the HPLC method for the determination of naringenin content in ECG and the method is accurate and reproducible. Naringenin content in varieties of ECG is stable and the difference of naringenin content between Citrus grandis Osbeck. Var. tomentosa Hort. and Citrus grandis (L.) Osbeck is not significant.

Random Amplified Polymorphic DNA Analysis of Germplasm Resources of Exocarpium Citri Grandis / 广州中医药大学学报

Objective To investigate the influence of producing areas and sexual reproduction on the genetic features of Citrus grandis‘Tomentosa’. Methods The genetic distance in different species was counted,and the DNA fingerprint of germplasm resources of Citrus grandis ‘Tomentosa’ was established after random amplified polymorphic DNA analysis of Citrus grandis ‘Tomentosa’ and Citrus grandis (L.) Osbeck of various species from different producing areas. Results The genetic distance index D of Citrus grandis ‘Tomentosa’ and Citrus grandis (L.) Osbeck of various species from different producing areas was in the range of 0.01~0.64. Conclusion Sexual reproduction and the changes of producing areas can result in the genetic diversity of Citrus grandis ‘Tomentosa’.

Assessment of the Quality of Two Different Species of Exocarpium Citri Grandis / 广州中医药大学学报

[Objective] To assess the quality of Citrus grandis Osbeck. Var. tomentosa Hort. (CGO Var. TH) and Citrus grandis (L. ) Osbeck (CGO). [Methods] The macroscopic feature and microscopic structure of CGO Var. TH and CGO were compared. Their content of total flavones (TF) was determined by spectrometry, naringin content and fingerprint by high performance liquid chromatography (HPLC) and the content of rhoifolin by thin-layer chromatography (TLC). Their expectorart, anti-inflammatory and antitussive effects were observed by phenol red test, auricular swelling test and ammonia-water-induced cough method. [Results] Obvious differences such as villi in the exocarp and non-grandular hair in the stem existed in the macroscopic feature and microscopic structure of CGO Var. TH and CGO. The contents o{ total flavones and naringin in CGO Var. TH were markedly higher than that in CGO and the rhoifolin content in CGO Var. TH was ten times as much as that in CGO. Markedly difference was also found in their fingerprint. The expectorant, anti-inflammatory and antitussive effects in CGO Var. TH were better than those in CGO. [Conclusion] The quality of CGO Var. TH is much better than that of CGO.

Determination of Naringin in Xingsu Ke Oral Liquid by High Performance Liquid Chromatography / 广州中医药大学学报

[Objective] To establish a determination method for naringin in Xingsu Ke Oral Liquid by high performance liquid chromatography (HPLC) . [ Methods ] At room temperature, HPLC was performed on ODS column; the mobile phase consisted of acetonitrile and 8% acetic acid (15: 85) and the detection wavelength was at 283nm. [Results] The linearity of naringin was good in the range of 1.6 - 9.6 mg/L ( r = 0.999 9), the average recovery was 98.62 % and RSD = 0.754 ( n = 5). [ Conclusion ] This method is simple and effective and can be used for the quality control of Xingsu Ke Oral Liquid.

Determination of the Rhoifolin in Exocarpium Citri Grandis By RP-HPLC / 中药新药与临床药理

Objective To establish a method for the determination of Rhoifolin content in Exocarpium citri grandis (ECG). Methods The RP-HPLC method was operated on MACHEREY-NAGEL 100-5 C_(18), with methanol and wateracetic acid (61: 4) as mobile phase, gradient elution, flow rate at 1.0 mL/min, and detection wavelength at 345nm. Results In the range from 0.816 ?g to 13. 056?g, Rhoifolin is in a good linearity with chromatographic peaks area, r=0.9995, the average recovery ratio is 101.064%, RSD=3.10%(n=5). The content of Rhoifolin in Tomentulosus Exocarpium Citri Grandis(TECG) ranged from 1.33% to 0.177% and that in Glabrous Exocarpium Citri Grandis (GECG) is less than 0.17%. Conclusions RP-HPLC method is with accuracy and a good reproducibility for the determination of the Rhoifolin content in ECG; the difference of Rhoifolin content in TECG and GECG is significant.

HPLC fingerprint of flavonoids in Exocarpium Citri Grandis / 中草药

Object To establish the HPLC fingerprint of flavonoids in Exocarpium Citri Grandis (ECG) Methods HPLC fingerprint of ten samples were established. The naringin was used as standard. The relative migration time and the percentage of peak area were used as parameters Results More than 30 peaks occurred on the HPLC fingerprint of flavonoids of ECG. The fingerprints of C. grandis ‘Tomentosa’ and C. grands had obivous differences Conclusion The method is simple and accurate with a good reproducibility. It could be used for quality control and identification of ECG

Influence of Different Processing Technique on the Content of Naringin in Exocarpium Citri Grandis / 中药新药与临床药理

Objective To assess the influence of different processing technique on the quality of Exocarpium citri grandis.Methods The RP-HPLC method was adopted to determine the naringin content in Exocarpium citri grandis.The chromatographic conditions were: Kromasil ODS column(250 mm?4.6 mm),mobile phase being methanol-water-acetic acid(35∶61∶4),flow rate 1.0 mL/min,detection wavelength at 283nm and column temperature at 25 ℃.Results There were obvious differences in the naringin content of Exocarpium citri grandis which was processed by different methods: the naringin content processed by electro static drying method was much higher than that by bake drying method, natural drying method and vacuum drying method.Conclusion The electro static drying method is the best way for drying Exocarpium citri grandis.

Effects of Fruit-age on Chemical Com position in Exocarpium Citri Grandis / 中药新药与临床药理

Objective To determine the appropriate collecting -time of Exocarpium citri grandis,the chemical compo-sition in Exocarpium citri grandis in different fruit -ages was compare d and analyzed.Methods Contents of naringein and bergapten were compared by TLC an d the total dihydroflavones in Exocarpium citri grandis was determined by spec-trophotometmic analysis with detection wavelength at 384nm.Results With the increase of fruit -age,the c ontents of naringein and total dihydroflavones in Exocarpium citri grandis decreased and the content of bergapt en increased.Con-clusion Fruit -age has an obviously effect on the chemical composition in Exocarpium citri grandis and collecting -time can influence the quality of medicin e materials.Further studies of Exocarpium citri grandis should be combined pharma-codynamics for fixing collecting -t ime.

Determination of isoimperatorin in Exocarpium citri grandis from various species and origins by TLC-Scanning / 中成药

Objective: To determinate the content of isoimperatorin in Exocarpium citri grandis from 10 various species and origins. Methods: The isoimperatorin was determinated at 305nm by TLC Scanning. Results: The content of isoimperatorin in Exocarpium citri grandis was very low, and the law of distribution could not be found. Conclusion: Although the content of isoimperatorin in Exocarpium citri grandis was very low, the method is very sensitive and simple and the result is accurate.