Mechanism of Nicotiflorin in San-Ye-Qing rhizome for anti-inflammatory effect in ulcerative colitis.

BACKGROUND: The incidence of ulcerative colitis (UC) is on the rise globally and the development of drugs targeting UC is urgent. Finding the target of action of natural products is important for drug discovery, elucidation of drug action mechanism, and disease mechanism. San-Ye-Qing (SYQ), is an ancient herbal medicine, but whether the powder of its rhizome has pharmacological effects against UC and its mechanism of action are not clear. PURPOSE: To evaluate the therapeutic effectiveness of rhizome powder of SYQ in treating UC, and conduct an isolation and characterization of the chemical constituents of the powder. Further, screen the most potent compounds among them and determine the potential mechanism for treating UC. METHODS: In vivo, the therapeutic effect of SYQ's rhizome powder on UC was assessed by mice's body weight, DAI score, colon length, tissue MPO activity, serum inflammatory markers, etc. Additionally, HPLC was used to isolate and identify the specific chemical components of SYQ's rhizome powder. Then, the most effective compounds and their therapeutic targets were analysed and screened in SYQ rhizome powder using network pharmacology, combined with CCK-8 assay, NO release assay and molecular docking assay, in conjunction with CETSA, DARTS, SPR and enzyme activity assay. Finally, the biological effects of the key compound on the targets were validated using Western blot and ELISA. RESULTS: In vivo, SYQ rhizome powder effectively restored mice's body weight, lowered DAI and pathological score, downregulated the expression of inflammatory biomarkers, and restored colon length, as well as the colonic epithelial and mucus barriers. Afterward, 9 compounds were isolated and identified from the powder of the rhizomes of SYQ by HPLC. Nicotiflorin is the primary compound in SYQ with the highest concentration. According to both CCK-8 and NO release tests, Nicotiflorin is also the most efficacious compound. Combined with network pharmacological prediction, molecular docking analysis, CETSA, DARTS, SPR and enzyme activity assay, Nicotiflorin may ultimately suppress inflammation by targeting p65 and inhibiting the NF-κB pathway, thereby attenuating the activation of NLRP3 inflammasome. To verify this conclusion, Western blot and ELISA experiments were conducted. CONCLUSIONS: Our results suggest that the extract from SYQ rhizomes has therapeutic properties for UC. Its active ingredient Nicotiflorin exerted potent anti-UC effects by binding to p65 and inhibiting the activation of NF-κB and NLRP3 inflammasomes.

Kaempferol and nicotiflorin ameliorated alcohol-induced liver injury in mice by miR-138-5p/SIRT1/FXR and gut microbiota.

Aims: Excessive alcohol consumption can lead to alcoholic liver diseases (ALDs). Tetrastigma hemsleyanum Diels et Gilg is a rare Chinese medicinal herb. Tetrastigma hemsleyanum Diels et Gilg has been validated to be highly effective for treating hepatitis. Kaempferol and nicotiflorin are two highly representative flavonoids, which have exhibit therapeutic effects on liver disease. Therefore, the protective mechanism of kaempferol and nicotiflorin on alcohol-induced liver injury were investigated. Main methods: Forty mice were used in this study. After treatment of Kaempferol and nicotiflorin, serum and liver were collected and used for determination of biochemical indicators, H&E staining, and molecular detection. The interaction of miRNAs from serum extracellular vehicles (EVs) with mRNAs and 16S rRNA sequencing of gut microbiota were also investigated. Key findings: The results showed that kaempferol and nicotiflorins significantly ameliorated alcohol-induced liver damage and observably regulated gut microbiota. Specifically, the levels of malondialdehyde (MDA) and CYP2E1 in the liver significantly reduced, and the activity of superoxide dismutase (SOD) and glutathione (GSH) in the liver evidently increased. They also significantly relieved liver oxidative stress and lipid accumulation by suppressing miR-138-5p expression, inversely enhancing deacetylase silencing information regulator 2 related enzyme-1 (SIRT1) levels and then decreasing farnesoid X receptor (FXR) acetylation, which then modulated Nrf2 and SREBP-1c signaling pathways to regulate oxidative stress and lipid metabolism induced by alcohol. Significance: Kaempferol and nicotiflorin reduced alcohol-induced liver damage by enhancing alcohol metabolism and reducing oxidative stress and lipid metabolism. The intestinal microorganism disorder was also ameliorated after oral kaempferol and nicotiflorin.

Ethnopharmacology of Rubus idaeus Linnaeus: A critical review on ethnobotany, processing methods, phytochemicals, pharmacology and quality control.

ETHNOPHARMACOLOGICAL RELEVANCE: Rubus idaeus Linnaeus (RI) is a Chinese herbal medicine that has been widely used in China for a long time to reinforce the kidney, nourish the liver, improve vision, and arrest polyuria. AIM OF THE STUDY: This work aims to evaluate the recent progress of the chemical composition, pharmacological activity, pharmacokinetics, metabolism, and quality control and of Rubus idaeus, which focuses on the insufficiency of existing research and will shed light on future studies of Rubus idaeus. METHODS: Literatures about "Rubus idaeus","Red raspberry" and "Fupenzi"are retrieved by browsing the database, such as Web of Science (http://www.webofknowledge.com/wos), Pubmed (https://pubmed.ncbi.nlm.nih.gov/), CNKI (http://www.cnki.net/), and Wanfang Data (http://www.wanfangdata.com.cn). In addition, related textbooks and digital documents are interrogated to provide a holistic and critical review of the topic. The period of the literature covered from 1981 to 2022. RESULTS: Approximately 194 compounds have been isolated from Rubus idaeus, which is rich in phenols, terpenoids, alkaloids, steroids, and fatty acids. Numerous investigations have demonstrated that Rubus idaeus exhibits many pharmacological activities, including hypoglycemic and hypolipidemic, anti-Alzheimer effect, anti-osteoporosis, hepatoprotective, anti-cancer, neuroprotective, anti-bacteria and skin care, etc. However, it is worth noting that most of the research is not associated with the conventional effect, such as reducing urination and treating opacity of the cornea. CONCLUSION: The effectiveness of Rubus idaeus has been proved by its long-term clinical application. The research on the pharmacological activity of Rubus idaeus has flourished. In many pharmacological experiments, only the high-dose group can achieve the corresponding efficacy, so the efficacy of Rubus idaeus needs to be further interrogated. Meanwhile, the relationship between pharmacological activity and specific compounds of Rubus idaeus has not been clarified yet. Last but not least, studies involving toxicology and pharmacokinetics are very limited. Knowledge of bioavailability and toxicological behavior of Rubus idaeus can help understand the herb's pharmacodynamic and safety profile.

Inhibitory effect of flavonoid glycosides on digestive enzymes: In silico, in vitro, and in vivo studies.

Flavonoid glycosides (FGs) appear to be good candidates for controlling blood glucose levels, so regular consumption of vegetables/fruits rich in FGs may prevent the consequences of type 2 diabetes (DM). Inhibition of digestive enzymes using natural FGs is a suitable dietary tool to regulate the hydrolysis of polysaccharides and overcome hyperglycemia. The aim of the current research is to find FGs that can effectively inhibit the digestive enzymes α-glucosidase (α-Gl) and α-amylase (α-Am). Accordingly, twenty-three FGs were selected and filtered through docking-based virtual screening. Based on the molecular docking and molecular dynamics (MD) simulation, among the 23 selected FGs, nicotiflorin and swertisin significantly inhibited α-Gl and α-Am, respectively. In vitro analysis revealed the inhibitory capacity of nicotiflorin on α-Gl was equal to IC50 at 0.148 mg/ml and the inhibitory activity of swertisin on α-Am was equal to IC50 at 1.894 mg/ml. It was found that nicotiflorin and swertisin act much like as a competitive inhibitor on α-Gl and α-Am, respectively. Furthermore, the fluorescence intensity of both enzymes decreased after interaction with two FGs. FT-IR and scanning electron microscopy (SEM) measurements suggested that the interactions could alter the conformation and microenvironment of the enzymes. Moreover, in vivo evaluation showed that the administration of nicotiflorin and swertisin can alleviate the blood glucose level of rats compared to the starch group (p < 0.05). The findings highlight that nicotiflorin and swertisin can be considered as possible inhibitors in treating diabetes mellitus via digestive enzymes inhibition.

Medicinal Importance, Pharmacological Activities and Analytical Aspects of a Flavonoid Glycoside 'Nicotiflorin' in the Medicine.

BACKGROUND: Herbal products are derived from different natural sources, mainly used as a source of food material and medicine in the health sectors since ancient times. Herbal products have gained popularity in modern medicine due to their beneficial health properties and pharmacological activities. Flavonoids are an important class of secondary metabolites found to be present in medicinal plants and their derived products. Flavonoids have been known for their anti-allergic, anti-bacterial, anti-diabetic, anti-inflammatory, anti-viral, anti-proliferative, anti-mutagenic, antithrombotic, anti-carcinogenic, anti-oxidant and hepatoprotective activities in the medicine. Nicotiflorin is a flavonoidal class phytochemical, found in medicinal plants, including Traditional Chinese medicine. METHODS: Scientific data on the medicinal importance and pharmacological activities of nicotiflorin have been collected and analyzed in the present work in order to know the therapeutic importance of nicotiflorin in medicine. Scientific data have been collected from Google, Google Scholar, Science Direct, PubMed and Scopus and analyzed in the present work. Analytical techniques data of separation, isolation and identification of nicotiflorin have also been collected and presented in the current work. Further biological importance of flavonoidal class phytochemicals was also discussed in the present work to understand the biological importance of nicotiflorin in medicine as it belongs to the flavonoid class. RESULTS: Scientific data analysis revealed the therapeutic importance and pharmacological activities of nicotiflorin. Nicotiflorin has significant biological potential against coronavirus, ischemia, renal impairment, hepatic complication, memory dysfunction and myocardial infarction. The biological potential of nicotiflorin against α-glucosidase and α-amylase enzymes, multiple myeloma cells and insulin secretion has also been discussed in the present work. Analytical data revealed the significance of modern analytical tools in medicine for the isolation, separation and quantification of nicotiflorin. CONCLUSION: Scientific data analysis of different research works revealed the biological importance and therapeutic potential of nicotiflorin in medicine.

Long-term protective effects of the nicotiflorin on ischemic stroke rats / 药学实践杂志

Objective To investigate the long-term protective effects of the nicotiflorin on ischemic stroke rats. Methods Ischemic stroke model in rats was established for this study. The effects of nicotiflorin on long-term survival rate, nervous system function, body weight and brain neurons in rats were observed. Results The nicotiflorin had significantly improved the long-term survival rate of cerebral ischemia rats, which also promoted weight gain, alleviated pathological damage of brain tissue, maintained morphology of brain neurons and function of nervous system. Conclusion The nicotiflorin has obvious long-term protective effect on ischemic stroke rats and the mechanism may be related to the protection of the structure and function of brain neurons.

Autophagy is involved in the neuroprotective effect of nicotiflorin.

ETHNOPHARMACOLOGICAL RELEVANCE: Nicotiflorin is a flavonoid glycoside derived from the traditional Chinese medicine FlosCarthami, dried petals of Carthamus tinctorius L., and has been confirmed to be a promising novel drug candidate for ischemic stroke. Yet, the exact role of nicotiflorin in cerebral I/R injury is uncharacterized and the possible mechanisms have not been clearly expounded. AIM OF THE STUDY: The present study was designed to determine the effect of nicotiflorin on cerebral ischemia/reperfusion (I/R) injury and its relationship with autophagy. MATERIALS AND METHODS: Middle cerebral artery occlusion (MCAO) in rats and oxygen-glucose deprivation and reintroduction (OGD/R) in SH-SY5Y cells were established in in vivo and in vitro models, respectively. The severity of MCAO was assessed by brain infarct size, neurological scores and survival rate. The severity of OGD/R was evaluated by cell viability, lactate dehydrogenase (LDH) release and cell apoptosis. The level of autophagy was evaluated both in vivo and in vitro. Autophagosomes were observed using transmission electron microscopy and autophagic flux was measured using mRFP-GFP-tandem fluorescent LC3 adenovirus. Autophagy-related proteins (LC3-II/I, SQSTM1, beclin-1, Phospho-mTOR/mTOR) were measured by immunoblot. Autophagy-related mRNA levels (Becn1, Atg7) were detected by Real-Time PCR. Inhibition of autophagy was implemented by 3-Methyladenine (3-MA) or chloroquine in vitro. RESULTS: In vivo, nicotiflorin treatment alleviated brain damage and neurological deficit while it dramatically increased 72 h survival rate in rats. In vitro, nicotiflorin treatment also ameliorated the severity of OGD/R. Moreover, nicotiflorin treatment increased ischemic penumbra autophagy (autophagosomes, BECN1, LC3-II/I ratio, SQSTM1, Phospho-mTOR/mTOR, Atg7). In vitro, nicotiflorin likewise enhanced autophagy and promoted autophagy flux. Furthermore, the blockade of autophagy by 3-MA or chloroquine disabled the efficacic of nicotiflorin in preventing cell damage upon OGD/R insult. CONCLUSION: These findings suggest that autophagy plays a significant role in the protective effect of nicotiflorin against ischemic stroke.

Mechanism of Action of Nicotiflorin from Tricyrtis maculata in the Treatment of Acute Myocardial Infarction: From Network Pharmacology to Experimental Pharmacology.

PURPOSE: Acute myocardial infarction (AMI) is a cardiovascular disease with a high fatality rate. In this study, we combined network pharmacology and experimental pharmacology and discovered the potential mechanism of action and the active ingredients of the lily, Tricyrtis maculata was discovered. The monomer compound with stronger activity was discovered through in vitro cell experiments. METHODS: Forty known compounds were isolated from T. maculata. Using TCMSP, Swiss Target Prediction, metaTarFisher, GeneCards and OMIM databases, targets of drug compositions and AMI-related genes were obtained, and the differential expression genes between AMI and normal tissues were extracted through the GEO database. Then, through an online mapping tool, the intersection genes were obtained to predict the possible effective components of T. maculata that can be used to treat AMI. The top five targets were selected for molecular docking via the protein-protein interaction (PPI) network to verify the binding activity between key compounds and target proteins. GO and KEGG enrichment analyses of the intersection genes were carried out with the program R to further screen key genes and effective compositions. On this basis, the compound with more optimal activity was screened and validated in vitro. RESULTS: In this study, 40 known monomer components were selected, and 1112 predicted genes, 1655 disease genes, 1425 differentially expressed genes, 1206 GO functions and 127 KEGG pathways were obtained. The results of molecular docking showed that the binding of MMP9 with drug components is stable. Through the comprehensive research of network pharmacology and experimental pharmacology, it was shown that T. maculata intervenes in the process of AMI through multicomponent, multitarget, and multichannel synergistic effects. It is speculated that the anti-AMI effect may be related to the regulation of the Akt/FoxO/BCl signaling pathway. Cellular experiments showed that nicotiflorin has satisfactory anti-inflammatory activity and endothelial protection and can reduce the release of nitric oxide (NO), an inflammatory medium after endothelial cell damage. CONCLUSION: This study reveals the therapeutic effect and relative mechanism of extract of T. maculata extract on AMI. Analysis revealed that nicotiflorin from T. maculata is a compound with satisfactory anti-inflammatory activity and endothelial protection, which provides a new direction and treatment basis for further experimental exploration and clinical treatment.

Molecular Docking Studies of Bioactive Nicotiflorin against 6W63 Novel Coronavirus 2019 (COVID-19).

BACKGROUND: COVID-19 which is known as the novel coronavirus was reported in December 2019 in Wuhan city, China and many people have been contaminated by environmental contamination and transmission from one human to another until now. OBJECTIVE: The objective of the present work is to establish the inhibitory potential of nicotiflorin, a Kaempferol 3-O-rutinoside flavonoid, against the deadly coronavirus (COVID-19) 6W63 (main protease 3Clpro protein), using molecular docking approach. METHODS: The Molegro Virtual Docker software (MVD) with a 30 Å grid resolution was used. The structure was drawn by Chem 3D software and energy minimization was done by the MM2 force field. The protein 6W63 was downloaded from the protein data bank. Molegro modeller was used for score calculations. RESULT: The molecular docking studies were carried out on nicotiflorin and standard inhibitor X77, where standard inhibitor was observed in a co-crystallized state with main protease 3Clpro protein 6W63. The MolDock score, Rerank Sore, and H Bond score of nicotiflorin and standard inhibitor X77 were observed as -173.058, -127.302, -21.9398 and -156.913,-121.296,-5.7369, respectively. CONCLUSION: Molecular docking studies have confirmed that the affinity of flavonoid nicotiflorin with the amino acids of the viral protein 6W63 was relatively more than the standard X77. For the effective treatment of novel coronavirus COVID-19, the effectiveness of the identified flavonoid nicotiflorin can further be evaluated for safety and efficacy parameters at both preclinical and clinical stages.

Nicotiflorin attenuates cell apoptosis in renal ischemia-reperfusion injury through activating transcription factor 3.

INTRODUCTION: Nicotiflorin is the main characteristic component of Nymphaea candida, which is a natural product that reportedly ameliorates acute injury of the liver and cerebral cortex, but the effect of nicotiflorin on acute kidney injury (AKI) remains unknown. This study aimed to investigate the effects of nicotiflorin on ischaemia/reperfusion (I/R) AKI and the associated mechanisms. METHODS: We performed both (a) in vivo experiments with C57BL/6 mice with bilateral renal pedicles clamped for 45 minutes and (b) in vitro experiments with human kidney epithelial cells (HK-2) exposed to hypoxia/reoxygenation to mimic I/R injury to study the role of nicotiflorin in AKI. RESULTS: In vivo, nicotiflorin administration exerted protective effects on renal injury, as demonstrated by reductions in the levels of caspase3 and Bad (P < .05), the upregulation of Bcl-2 expression (P < .05) and improved renal histologic changes, which suggested that nicotiflorin can alleviate I/R injury and cell apoptosis. In vitro, nicotiflorin at a concentration of 75 µg/mL protected cells from hypoxia, which further confirmed that nicotiflorin exerts beneficial effects on hypoxia/reoxygenation. Through computational molecular docking, we found that activating transcription factor 3 (ATF3) exhibits a robust interaction with nicotiflorin with a simulated binding energy of -9.2°. We verified the interaction of nicotiflorin with ATF3 in HK-2 cells, and found that nicotiflorin reduced the apoptosis of HK-2 through ATF3. CONCLUSION: Based on the above-described results, nicotiflorin appears to have a beneficial impact on deteriorated renal function, as demonstrated using an experimental I/R model. The underlying mechanisms of nicotiflorin might inhibit HK-2 cell apoptosis through ATF3.

Flavonoid glycosides and their putative human metabolites as potential inhibitors of the SARS-CoV-2 main protease (Mpro) and RNA-dependent RNA polymerase (RdRp)

BACKGROUND Since the World Health Organization (WHO) declared Coronavirus disease 2019 (COVID-19) to be a pandemic infection, important severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) non-structural proteins (nsp) have been analysed as promising targets in virtual screening approaches. Among these proteins, 3-chymotrypsin-like cysteine protease (3CLpro), also named main protease, and the RNA-dependent RNA polymerase (RdRp), have been identified as fundamental targets due to its importance in the viral replication stages. OBJECTIVES To investigate, in silico, two of the most abundant flavonoid glycosides from Dysphania ambrosioides; a medicinal plant found in many regions of the world, along with some of the putative derivatives of these flavonoid glycosides in the human organism as potential inhibitors of the SARS-CoV-2 3CLpro and RdRp. METHODS Using a molecular docking approach, the interactions and the binding affinity with SARS-CoV-2 3CLpro and RdRp were predicted for quercetin-3-O-rutinoside (rutin), kaempferol-3-O-rutinoside (nicotiflorin) and some of their glucuronide and sulfate derivatives. FINDINGS Docking analysis, based on the crystal structure of 3CLpro and RdRp, indicated rutin, nicotiflorin, and their glucuronide and sulfate derivatives as potential inhibitors for both proteins. Also, the importance of the hydrogen bond and π-based interactions was evidenced for the presumed active sites. MAIN CONCLUSIONS Overall, these results suggest that both flavonoid glycosides and their putative human metabolites can play a key role as inhibitors of the SARS-CoV-2 3CLpro and RdRp. Obviously, further researches, mainly in vitro and in vivo experiments, are necessary to certify the docking results reported here, as well as the adequate application of these substances. Furthermore, it is necessary to investigate the risks of D. ambrosioides as a phytomedicine for use against COVID-19.

Inhibition of cerebral ischemia/reperfusion injury-induced apoptosis: nicotiflorin and JAK2/STAT3 pathway.

Nicotiflorin is a flavonoid extracted from Carthamus tinctorius. Previous studies have shown its cerebral protective effect, but the mechanism is undefined. In this study, we aimed to determine whether nicotiflorin protects against cerebral ischemia/reperfusion injury-induced apoptosis through the JAK2/STAT3 pathway. The cerebral ischemia/reperfusion injury model was established by middle cerebral artery occlusion/reperfusion. Nicotiflorin (10 mg/kg) was administered by tail vein injection. Cell apoptosis in the ischemic cerebral cortex was examined by hematoxylin-eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Bcl-2 and Bax expression levels in ischemic cerebral cortex were examined by immunohistochemial staining. Additionally, p-JAK2, p-STAT3, Bcl-2, Bax, and caspase-3 levels in ischemic cerebral cortex were examined by western blot assay. Nicotiflorin altered the shape and structure of injured neurons, decreased the number of apoptotic cells, down-regulates expression of p-JAK2, p-STAT3, caspase-3, and Bax, decreased Bax immunoredactivity, and increased Bcl-2 protein expression and immunoreactivity. These results suggest that nicotiflorin protects against cerebral ischemia/reperfusion injury-induced apoptosis via the JAK2/STAT3 pathway.

Hepatoprotective Effects of Nicotiflorin from Nymphaea candida against Concanavalin A-Induced and D-Galactosamine-Induced Liver Injury in Mice.

Nymphaea candida was used to treat hepatitis in Ugyhur medicine, and nicotiflorin (kaempferol 3-O-ß-rutinoside) is the main characteristic component in this plant [...].

Anxiolytic-like effects and toxicological studies of Brickellia cavanillesii (Cass.) A. Gray in experimental mice models.

ETHNOPHARMACOLOGICAL RELEVANCE: Brickellia cavanillesii (Asteraceae) (Cass.) A. Gray is one of the popular plants consumed in Central America and Mexico for the treatment of several diseases such as hypertension, diabetes and anxiety, among others. AIM OF THE STUDY: To determine the anxiolytic-like effect of B. Cavanillesii and the safety of its use through toxicological studies. MATERIAL AND METHODS: Anxiolytic-like effects of soluble-methanol extract of B. cavanillesii (MEBc) were evaluated in ambulatory activity (open-field test), hole-board test, cylinder of exploration, the elevated plus-maze and the potentiation of the sodium pentobarbital-induced hypnosis mice models. On the other hand, in vivo toxicological studies were conducted on acute and sub-acute mice models recommended by OECD. Active MEBc was subjected to phytochemical studies through conventional chromatographic techniques to isolate bioactive compounds. RESULTS: MEBc (100mg/Kg) showed significant anxiolytic-like effect on animal model used (p<0.05). The phytochemical analysis of MEBc allowed the isolation of two major compounds nicotiflorin and acacetin, among others. Both compounds were found to be partially responsible for the anxiolytic-like effects. Moreover, a median lethal dose (LD50) higher than 2000mg/Kg was determined in mice and sub-acute oral administration of MEBc (100mg/Kg) did not alter body weight, clinical chemistry parameters (ALT and AST) and it did not induce any toxic nor alteration in the liver, kidney and heart functions. CONCLUSIONS: In current investigation, we have shown that MEBc has a wide range of pharmacology-toxicology patterns. The results support further investigation of MEBc as a potential anxiolytic phytomedicinal agent.

Changes of major tea polyphenols and production of four new B-ring fission metabolites of catechins from post-fermented Jing-Wei Fu brick tea.

HPLC analysis of samples from four major fermentation procedures of Jing-Wei Fu brick tea showed that the level of major tea catechins epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) dropped increasingly to about 1/3 in the final product. Phytochemical study of the final product led to the discovery of four new B-ring fission metabolites of catechins (BRFCs) Fuzhuanin C-F (1-4) together with three known BRFCs (5-7), six known catechins (8-13), five simple phenols (14-18), seven flavones and flavone glycosides (19-25), two alkaloids (26, 27), three triterpenoids (28-30) and one steroid (31). The structures were elucidated by spectroscopic methods including 1D and 2D NMR, LC-HR-ESI-MS, IR, and CD spectra. Five compounds (16-18, 28, 29) were reported for the first time in tea. Possible pathways for the degradation of major tea catechins and the generation of BRFCs were also provided.