Fulvanines J-K, two rare lactam pyrrole alkaloids from Hemerocallis fulva.

Two rare jatropham lactam derivatives, named as fulvanines J-K (1-2), together with six known pyrrole alkaloids, 5,5'-oxydi(3-methyl-3-pyrrolin-2-one) (3), (-)-5-hydroxy-3-methyl-3-pyrrolin-2-one (jatropham) (4), (±)-5-O-methyljatropham (5), perlolyrine (6), butyl-2-formyl-5-(hydroxymethyl)-1H-pyrrole-1-butanoate (7), and hemerocallisamine II (8), were isolated from the flower of Hemerocallis fulva. Their structures were elucidated on the basis of spectroscopic methods and compared with the NMR spectra data in the literature. All compounds were evaluated for their anti-complementary activity in vitro, and compounds 1, 4, and 6 exhibited anti-complement effect with CH50 values from 0.61 to 1.42 mM.

Daylily (Hemerocallis fulva Linn.) flowers improve sleep quality in human and reduce nitric oxide and interleukin-6 production in macrophages.

The flowers of daylily (Hemerocallis fulva Linn.) have been used as vegetable and medicinal herb for thousands of years in Taiwan and eastern Asia. Daylily flowers have been demonstrated to exert several biomedical properties. In this study, we provided the evidences show that daylily flowers exert anti-inflammatory activity in vitro and improved the sleep quality in vivo. We demonstrated that adult volunteers received water extract of daylily flowers improved sleep quality, sleep efficiency and daytime functioning, while sleep latency was reduced, compared to the adult volunteers received water. In addition, we demonstrated that aqueous and ethanol extracts of daylily flowers inhibited nitric oxide and interleukin-6 production in lipopolysaccharide-activated macrophages. Furthermore, the quantitative high performance liquid chromatography-based analysis showed the rutin content of the aqueous extract, ethanolic extract, ethyl acetate fractions of ethanolic extract, and water fractions of ethanolic extract were 7.27, 23.30, 14.71, and 57.43 ppm, respectively. These results indicate that daylily flowers have the potential to be a nutraceutical for improving inflammatory-related diseases and sleep quality in the future.

Freeze-dried powder of daylily bud improves bromocriptine-induced lactation disorder in rats via JAK2/STAT5 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Milk deficiency is a prevalent problem in the world. Daylily (Hemerocallis citrina Borani), called the Chinese mother flower, is a traditional vegetable and is believed to possess a galactagogue effect in China. Flavonoids and phenols are considered as the active ingredients of daylily to promote lactation and improve depression. AIM OF THE STUDY: The aim of this study was to investigate the prolactin effects of freeze-dried powder of flower buds of H. citrina Baroni in rat and its action mechanisms. MATERIALS AND METHODS: The chemical constituents of flower buds of H. citrina Baroni treated by different drying techniques were analyzed by ultrahigh pressure liquid chromatography-mass spectrometry. Sprague-Dawley (SD) rat model induced by bromocriptine was used to evaluate the effect of freeze-dried powder of daylily buds on promoting lactation. Network pharmacology method, ELISA, qPCR, and Western blot were used to clarify the action mechanisms. RESULTS: We detected 657 compounds in daylily buds. The relative contents of total flavonoids and phenols in freeze-dried samples were higher than those in dried ones. Bromocriptine, as a dopamine receptor agonist, can significantly inhibit prolactin in rats. Daylily buds can restore the levels of prolactin, progesterone and estradiol depressed by bromocriptine, effectively improve the milk production of the rat, and promote the repair of rat mammary gland tissue. We analyzed the relationship between the chemical components of daylily buds and the genes related to lactation with network pharmacology method, revealing that flavonoids and phenols may be the active components that promoted milk production via JAK2/STAT5 pathway, which was confirmed by the results of qPCR and Western blot. Daylily buds can increase the mRNA expression of PRLR, CSN2, LALBA and FASN and the protein expression of PRLR, JAK2 and STAT5. CONCLUSION: Daylily buds can improve the insufficient lactation of rats induced by bromocriptine through PRLR/JAK2/STAT5 pathway, and the freeze-dried processing method may better retain the active components of flavonoids and phenols that promote milk in daylily.

Study on Anti-Constipation Effects of Hemerocallis citrina Baroni through a Novel Strategy of Network Pharmacology Screening.

Daylily (Hemerocallis citrina Baroni) is an edible plant widely distributed worldwide, especially in Asia. It has traditionally been considered a potential anti-constipation vegetable. This study aimed to investigate the anti-constipation effects of daylily from the perspective of gastro-intestinal transit, defecation parameters, short-chain organic acids, gut microbiome, transcriptomes and network pharmacology. The results show that dried daylily (DHC) intake accelerated the defecation frequency of mice, while it did not significantly alter the levels of short-chain organic acids in the cecum. The 16S rRNA sequencing showed that DHC elevated the abundance of Akkermansia, Bifidobacterium and Flavonifractor, while it reduced the level of pathogens (such as Helicobacter and Vibrio). Furthermore, a transcriptomics analysis revealed 736 differentially expressed genes (DEGs) after DHC treatment, which are mainly enriched in the olfactory transduction pathway. The integration of transcriptomes and network pharmacology revealed seven overlapping targets (Alb, Drd2, Igf2, Pon1, Tshr, Mc2r and Nalcn). A qPCR analysis further showed that DHC reduced the expression of Alb, Pon1 and Cnr1 in the colon of constipated mice. Our findings provide a novel insight into the anti-constipation effects of DHC.

Steamed daylily flower (Hemerocallis fulva L.) protected cardiac and hepatic cells against ethanol induced apoptotic and oxidative damage.

Our previous study examined the phytochemical composition and bio-activities of raw daylily flower (Hemerocallis fulva L.). However, this plant food is usually served via heat process such as cooking in a soup. This study aimed to investigate the phytochemical profile and biofunctions of steamed daylily flower (SDF). The content of total phenolic acids, total flavonoids, total carotenoids, total anthocyanins and total triterpenoids in SDF aqueous extract was assessed. Normal cardiac and hepatic cells, H9c2 and L-02 cells, were used to evaluate the protective effects of SDF against ethanol. SDF concentrations of 0.25%, 0.5%, and 1% were applied to treat H9c2 or L-02 cells for 48 h at 37 °C initially, followed by exposure to ethanol at 150 mM for 24 h at 37 °C. Results showed that the content of assessed phytochemicals was in the range of 1019-2045 mg/100 g dry weight. Flavonoids and triterpenoids were two major detected phytochemicals in SDF. SDF treatments at 0.5% and 1% increased the viability of H9c2 cells, but at three concentrations enhanced the survival of L-02 cells. SDF at 0.5% and 1% up-regulated Bcl-2 messenger RNA (mRNA) expression and down-regulated Bax mRNA expression. Ethanol increased reactive oxygen species production, decreased glutathione content, as well as lowered glutathione peroxidase and catalase activities. SDF treatments reversed these changes. SDF at 0.5% and 1% reduced the activity of cytochrome P450 2E1 and nicotinamide adenine dinucleotide phosphate oxidase, limited p47phox mRNA expression, as well as enhanced factor E2-related factor 2 and heme oxygenase-1 mRNA expression. SDF at three concentrations decreased gp91phox mRNA expression. In conclusion, these novel findings indicated that SDF aqueous extract was rich in phytochemicals and provided anti-apoptotic and anti-oxidative actions to protect cardiac and hepatic cells against ethanol. Thus, SDF might be considered as a functional food with multiple bio-activities.

An Investigation of the Anti-Depressive Properties of Phenylpropanoids and Flavonoids in Hemerocallis citrina Baroni.

The World Health Organization predicts that over the next several years, depression will become the most important mental health issue globally. Growing evidence shows that the flower buds of Hemerocallis citrina Baroni (H. citrina) possess antidepressant properties. In the search for new anti-depression drugs, a total of 15 phenylpropanoids and 22 flavonoids were isolated and identified based on spectral data (1D and 2D NMR, HR-ESI-MS, UV) from H. citrina. Among them, compound 8 was a novel compound, while compounds 1-4, 6, 9, 10, 15, 17, 24-26, 28, and 37 were isolated for the first time from Hemerocallis genus. To study the antidepressant activity of phenylpropanoids and flavonoids fractions from H. citrina, macroporous resin was used to enrich them under the guidance of UV characteristics. UHPLC-MS/MS was applied to identify the constituents of the enriched fractions. According to behavioral tests and biochemical analyses, it showed that phenylpropanoid and flavonoid fractions from H. citrina can improve the depressive-like mental state of chronic unpredictable mild stress (CUMS) rats. This might be accomplished by controlling the amounts of the inflammatory proteins IL-6, IL-1ß, and TNF-α in the hippocampus as well as corticosterone in the serum. Thus, the monomer compounds were tested for their anti-neuroinflammatory activity and their structure-activity relationship was discussed in further detail.

Polysaccharide from Hemerocallis citrina Borani by subcritical water: Bioactivity, purification, characterization, and anti-diabetic effects in T2DM rats.

Hemerocallis citrina Borani (daylily) has various health benefits. However, the structural characterization and hypoglycemic effects of its polysaccharide remain unclear. Here, we first report for the first time, the effects of subcritical water extraction temperature on bioactivity of Hemerocallis citrina Borani polysaccharide (HCBP). HCBP extracted at 160 °C had better scavenging ability of free-radical and pancreatic lipase inhibition. This study aimed to investigate the structural characterization and anti-diabetic effects of aforementioned HCBP. HCBP was separated into three fractions using a DEAE-Sepharose Fast Flow. HCBP-1 was the major component with lower molecular weight, HCBP-2 was the least abundant component, HCBP-3 was the component with higher uronic acid and molecular weight. HCBP treatment significantly decreased the concentrations of fasting blood glucose, insulin, total cholesterol, triglyceride, and low density lipoprotein, as well as improved glucose intolerance and insulin resistance in type 2 diabetes mellitus (T2DM) rats. HCBP also protected the kidneys and pancreatic organs by histopathology and immunohistochemical analyses in T2DM rats. In addition, HCBP significantly increased the antioxidant enzymes activities and decreased the level of malonaldehyde to alleviated the oxidative stress injury. The results indicate HCBP extracted by subcritical water is promising functional food ingredients and could be used in T2DM treatment.

Study on Extraction and Antioxidant Activity of Flavonoids from Hemerocallis fulva (Daylily) Leaves.

Hemerocallis fulva is a medical and edible plant. In this study, we optimized the ultrasound-assisted extraction (UAE) process of extracting flavonoids from Hemerocallis fulva leaves by single-factor experiments and response surface methodology (RSM). The optimum extraction conditions generating the maximal total flavonoids content was as follows: 70.6% ethanol concentration; 43.9:1 mL/g solvent to sample ratio; 61.7 °C extraction temperature. Under the optimized extraction conditions, the total flavonoid content (TFC) in eight Hemerocallis fulva varieties were determined, and H. fulva (L.) L. var. kwanso Regel had the highest TFC. The cytotoxicity of the extract was studied using the Cell Counting Kit-8 (CCK-8 assay). When the concentration was less than 1.25 mg/mL, the extract had no significant cytotoxicity to HaCaT cells. The antioxidant activity was measured via chemical antioxidant activity methods in vitro and via cellular antioxidant activity methods. The results indicated that the extract had a strong ABTS and •OH radical scavenging activity. Additionally, the extract had an excellent protective effect against H2O2-induced oxidative damage at a concentration of 1.25 mg/mL, which could effectively reduce the level of ROS to 106.681 ± 9.733% (p < 0.001), compared with the 163.995 ± 6.308% of the H2O2 group. We identified five flavonoids in the extracts using high-performance liquid chromatography (HPLC). Infrared spectroscopy indicated that the extract contained the structure of flavonoids. The results showed that the extract of Hemerocallis fulva leaves had excellent biocompatibility and antioxidant activity, and could be used as a cheap and potential source of antioxidants in the food, cosmetics, and medicine industries.

Exploring the Extraction Methods of Phenolic Compounds in Daylily (Hemerocallis citrina Baroni) and Its Antioxidant Activity.

Daylily is a valuable plant resource with various health benefits. Its main bioactive components are phenolic compounds. In this work, four extraction methods, ultrasonic-assisted water extraction (UW), ultrasonic-assisted ethanol extraction (UE), enzymatic-assisted water extraction (EW), and enzymatic-assisted ethanol extraction (EE), were applied to extract phenolic compounds from daylily. Among the four extracts, the UE extract exhibited the highest total phenolic content (130.05 mg/100 g DW) and the best antioxidant activity. For the UE extract, the DPPH value was 7.75 mg Trolox/g DW, the FRAP value was 14.54 mg Trolox/g DW, and the ABTS value was 15.37 mg Trolox/g DW. A total of 26 phenolic compounds were identified from the four extracts, and the UE extract exhibited a higher abundance range of phenolic compounds than the other three extracts. After multivariate statistical analysis, six differential compounds were selected and quantified, and the UE extract exhibited the highest contents of all six differential compounds. The results provided theoretical support for the extraction of phenolic compounds from daylily and the application of daylily as a functional food.

Hemecitones A and B: two phenanthrenes with cytotoxicity from Hemerocallis fulva (L.) L.

Two new phenanthrenes, Hemecitones A and B, were isolated from Hemerocallis fulva (L.) L. Their structures were determined by UV, IR, HR-ESI-MS, 1D and 2D NMR analysis. The in vitro cytotoxic activities of compounds 1 and 2 were studied against breast carcinoma MDA-MB-231, hepatocellular carcinoma HepG2 and lung carcinoma A549 cell lines. And compounds 1 and 2 exhibited anti-proliferative effects against these least one of the three types of cell lines with IC50 ranging from 12.57 ± 2.34 to 31.22 ± 1.36 µM.[Formula: see text].

Optimization of ultrasonic-assisted extraction of polysaccharides from Hemerocallis citrina and the antioxidant activity study.

The present study discussed the optimization of the ultrasonic-assisted extraction of polysaccharides from daylily polysaccharides (DPs). The extracted crude polysaccharides were further separated and purified, and the antioxidant activities including 1,1-diphenyl-2-111 picrylhydrazyl (DPPH) radical scavenging, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) radical scavenging, hydroxyl radical scavenging, and ferric-reducing antioxidant power (FRAP) activities of the obtained fractions were also evaluated. The results showed that the optimal ultrasonic-assisted extraction parameters with DPs yield of 15.25 ± 1.13% were water to powder ratio of 25 ml/g, extraction power of 694 W, extraction temperature of 71°C, extraction time of 38 min, and three times extraction. By DEAE Sepharose Fast Flow column, four water-soluble polysaccharide fractions (DP-1, DP-2, DP-3, and DP-4) were successfully obtained. Monosaccharide component analysis showed that the four obtained fractions were all hetero-polysaccharides that mainly contained rhamnose, arabinose, fructose, galactose, glucose, galacturonic acid, and glucuronic acid in different molar ratios. All the four DP fractions did show obvious antioxidant activities in vitro, and the DP-3 component had relatively high ABTS free radical scavenging activity. Overall, our research showed that DPs could provide cheap raw materials for the development of natural antioxidants in medicines, functional foods, and even cosmetics. PRACTICAL APPLICATION: This article deals with the optimization of the ultrasonic-assisted extraction of polysaccharides from daylily and its antioxidant activities. The results showed that the optimal ultrasonic-assisted extraction yield of DPs was 15.25 ± 1.13%. By DEAE Sepharose Fast Flow column, four water-soluble polysaccharide fractions were successfully obtained, and all the four DP fractions did show obvious antioxidant activities in vitro. Daylily polysaccharides could provide cheap raw materials for the development of natural antioxidants in medicines, functional foods, and even cosmetics.

Systematic identification metabolites of Hemerocallis citrina Borani by high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry combined with a screening method.

Hemerocallis citrina Borani is an important crop and its flower buds are widely consumed in East Asian areas as a vegetable, as well as in traditional Chinese medicine, due to its health-promoting properties. Metabolites present in plant-derived foods or medicines are in part responsible for their desirable flavor profiles and health benefits. Nevertheless, detailed information about these compounds in H. citrina is scarce. Therefore, this study aimed to investigate the metabolites by high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS). In this study, a total of 144 compounds, including 14 amides, 25 polyphenols, 44 flavonols, 35 anthraquinones, 15 naphthols, and 11 other components, were detected by the established screening method and were identified by their precise m/z values, characteristic tandem mass spectrometry (MS/MS) data and fragmentation pathways of references, 111 of which were reported in this plant for the first time. The distribution of identified ingredients in different parts of H. citrina was determined. Interestingly, colchicine, which had been reported as a toxic compound in the fresh flower buds in previous studies and various news reports, was not found. This work marks the first comprehensive study of metabolites from commercial flower buds and different parts of H. citrina.

Reveals of New Candidate Active Components in Hemerocallis Radix and Its Anti-Depression Action of Mechanism Based on Network Pharmacology Approach.

The global depression population is showing a significant increase. Hemerocallis fulva L. is a common Traditional Chinese Medicine (TCM). Its flower buds are known to have ability to clear away heat and dampness, detoxify, and relieve depression. Ancient TCM literature shows that its roots have a beneficial effect in calming the spirit and even the temper in order to reduce the feeling of melancholy. Therefore, it is inferred that the root of Hemerocallis fulva L. can be used as a therapeutic medicine for depression. This study aims to uncover the pharmacological mechanism of the antidepressant effect of Hemerocallis Radix (HR) through network pharmacology method. During the analysis, 11 active components were obtained and screened using ADME-absorption, distribution, metabolism, and excretion- method. Furthermore, 267 HR targets and 740 depressive disorder (DD) targets were gathered from various databases. Then protein-protein interaction (PPI) network of HR and DD targets were constructed and cluster analysis was applied to further explore the connection between the targets. In addition, gene ontology (GO) enrichment and pathway analysis was applied to further verify that the biological process related to the target protein is associated with the occurrence of depression disorder. In conclusion, the most important bioactive components-anthraquinone, kaempferol, and vanillic acid-can alleviate depression symptoms by regulating MAOA, MAOB, and ESR1. The proposed network pharmacology strategy provides an integrating method to explore the therapeutic mechanism of multi-component drugs on a systematic level.

Ethyl Acetate Fraction of Hemerocallis citrina Baroni Decreases Tert-butyl Hydroperoxide-Induced Oxidative Stress Damage in BRL-3A Cells.

The main purposes of this study were to screen the antioxidant activities of various fractions of Hemerocallis citrina Baroni and test their hepatoprotective effects in vitro. Antioxidant assays (2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and reducing power experiments) and tert-butyl hydroperoxide- (t-BHP-) induced BRL-3A oxidative damage experiments were performed in vitro. The H. citrina ethyl acetate fraction (HCEA) was determined to have strong antioxidant activity because of its high flavonoid and polyphenol content. Ultraperformance liquid chromatography- (UPLC-) photodiode array (PDA)/mass spectrometry (MS) analysis showed that the main components of the HCEA were flavonoids and caffeic acid derivatives. A total of 17 compounds were identified. HCEA also effectively protected the liver against t-BHP-induced oxidative stress injury and significantly reduced reactive oxygen (ROS) accumulation. Moreover, HCEA significantly reduced levels of alanine aminotransferase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH). Further studies have shown that HCEA inhibits t-BHP-induced apoptosis by increasing B-cell lymphoma-2 (BCL-2) activity and decreasing caspase-3 and caspase-9 activity. Moreover, HCEA enhanced the activity of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), as well as the total antioxidant capacity (T-AOC), and increased the antioxidant level of glutathione (GSH) in BRL-3A cells. HCEA increased the antioxidant capacity of cells by increasing the gene expression of AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (ERK), P38, nuclear factor, erythroid 2 like 2 (Nrf2), SOD, glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modifier subunit (GCLM), and heme oxygenase 1 (HO-1), which are associated with antioxidant pathways to protect against oxidative stress. In conclusion, HCEA protected BRL-3A cells against t-BHP-induced oxidative stress damage via antioxidant and antiapoptosis pathways. Therefore, H. citrina Baroni may serve as a potential hepatoprotective drug.

Aqueous and Ethanol Extracts of Daylily Flower (Hemerocallis fulva L.) Protect HUVE Cells Against High Glucose.

The content of several phenolic acids and flavonoids in aqueous extract (AE) and ethanol extract (EE) of daylily flower (Hemerocallis fulva L.) was analyzed. The effects of AE or EE at 0.5%, 1%, or 2% in HUVE cells against high glucose-induced cell death, oxidative, and inflammatory damage were examined. Results showed that seven phenolic acids and seven flavonoids could be detected in AE or EE, in the range of 29 to 205 and 41 to 273 mg/100 g, respectively. Compared with the control groups, high glucose raised the activity of caspase-3 and caspase-8; suppressed Bcl-2 mRNA expression and increased Bax mRNA expression; and induced HUVE cells apoptosis. The pretreatments from AE or EE at 1% or 2% reduced caspase-3 activity and Bax mRNA expression, and enhanced cell viability. High glucose decreased glutathione content; stimulated the production of reactive oxygen species, interleukin-6, tumor necrosis factor-alpha, and prostaglandin E2 ; raised the activity of cyclooxygenase-2 and nuclear factor kappa B p50/65 binding; and reduced the activity of glutathione peroxidase, glutathione reductase, and catalase in HUVE cells. AE pretreatments at 1% and 2% reversed these changes. These novel findings suggested that daylily flower was rich in phytochemicals, and could be viewed as a potent functional food against diabetes.

Study the effects of drying processes on chemical compositions in daylily flowers using flow injection mass spectrometric fingerprinting method and chemometrics.

Daylily flowers is an important vegetable in eastern Asia, widely used in many dishes. Daylily flowers are usually sold in dried forms in the supermarkets. There are a few method for processing fresh daylily flowers, however, no study has been conducted to analyze the effects of the processing methods in the composition of final products. In the present study, a flow-injection mass spectrometric fingerprinting (FIMS) method in combination with principal component analysis (PCA) was used to differentiate two species of daylily flowers (Mengzi and Chongli) with three different treatments (vacuum freeze drying, solar drying and hot-air drying treatments) for each. The results showed the fast and simple FIMS method could successfully differentiate between species (raw material) and treatments among each species. However, the two species could not be discriminated very well after being processed in hot-air drying treatment. The difference in chemical profiles of species and treatments were discussed.

Evaluation of the toxicological properties and anti-inflammatory mechanism of Hemerocallis citrina in LPS-induced depressive-like mice.

Hemerocallis citrina Baroni (Liliaceae), a Liliaceae plant, has been widely used in food and traditional medicine. This study investigated the safety of ethanol extracts from Hemerocallis citrina (HCE) after oral treatment (p.o.) and evaluating the anti-inflammatory mechanism of HCE in a lipopolysaccharide (LPS)-induced depressive-like model. First, in an 8-week experimental procedure, blood and tissue samples collected from mice were used for biochemical and histopathological analysis every two weeks. Neither the body weight nor relative organ weights were affected by HCE administration. Only the total cholesterol levels were decreased by HCE administration. Histopathological analysis showed no significant liver and kidney changes caused by HCE. In addition, in an LPS-induced mouse depressive-like model, HCE significantly reversed the reduction of sucrose preference with LPS. The results also indicated that LPS activated the nuclear factor-κB (NF-κB), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in the prefrontal cortex. In contrast, these activations were normalized by HCE pretreatment. In summary, our study provided essential evidence for the safety of Hemerocallis citrina in both food and medicine. The results also demonstrated that HCE exhibited antidepressant-like effects that might be related to inhibition of the NF-κB signaling pathway.

Eight new γ-lactam alkaloids from the roots of the Hemerocallis minor Mill.

Eight new γ-lactam alkaloids, hemerominors A-H (1-8), including two pair of epimers (1-4), together with six known compounds (9-14) were isolated from the roots of Hemerocallis minor Mill. The structures of 1-8 were established on the basis of extensive NMR studies and HR-MS measurements as well as comparison with literature data. The absolute configurations of 1-8 were determined by CD spectral analysis and modified Mosher's method. All of compounds were evaluated for their inhibitory effects against LPS-induced NO production in RAW264.7 macrophages. Among them, compound 13 exhibited moderate inhibitory activity against NO production and with IC50 value of 18.0 µM.

Daylily protein constituents of the pollen and stigma a proteomics approach.

This study was aimed at the identification and quantification of the protein components of the pollen grains in parallel with the distal stigmatic tissue of tetraploid cultivars. Proteomes were analyzed using iTRAQ 4plex labeling, peptides separation by online RP-nano-LC and analysis by ESI-MS/MS. Protein identification and quantification were made using the Asparagales database as a reference. A total of 524,037 MS/MS spectra were produced from pollen and stigma samples. From these, a total of 8368 peptides wereidentified corresponding to 994 unique peptides and 432 protein groups. Among them, 128 differentially expressed proteins were retained for further analysis. In absence of the daylily genome availability, we exploited numerous databases and bioinformatics resources to exploring the putative biological functions of these proteins. The profile of differentially expressed proteins suggests an important representation of functions associated to the signalling and response against endogenous and environmental stresses, including several enzymes implicated in the biosynthesis of antibiotics. The abundance in stigma of several structural proteins of the ribosomal sub-units as well as of the core histones suggest that the translation processes and the regulation of gene expression in stigma is a more active mechanism than in pollen. In addition, pollen prioritizes the synthesis of fructose and glucose as opposed to sucrose in stigma as a source of energy. Finally, the modulated proteins in Hemerocallis point to several pathways that give potential clues concerning the molecular mechanisms underlying the functions of the pollen and the stigmatic fluid in daylily reproduction.

Effects of phenolic constituents of daylily flowers on corticosterone- and glutamate-treated PC12 cells.

BACKGROUND: Daylily flowers, the flower and bud parts of Hemerocallis citrina or H. fulva, are well known as Wang-You-Cao in Chinese, meaning forget-one's sadness plant. However, the major types of active constituents responsible for the neurological effects remain unclear. This study was to examine the protective effects of hydroalcoholic extract and fractions and to identify the active fractions. METHODS: The extract of daylily flowers was separated with AB-8 resin into different fractions containing non-phenolic compounds, phenolic acid derivatives and flavonoids as determined using UPLC-DAD chromatograms. The neuroprotective activity was measured by evaluating the cell viability and lactate dehydrogenase release using PC12 cell damage models induced by corticosterone and glutamate. The neurological mechanisms were explored by determining their effect on the levels of dopamine (DA), 5-hydroxy tryptamine (5-HT), γ-aminobutyric acid (GABA), noradrenaline (NE) and acetylcholine (ACh) in the cell culture medium measured using an LC-MS/MS method. RESULTS: Pretreatment of PC12 cells with the extract and phenolic fractions of daylily flowers at concentrations ranging from 0.63 to 5 mg raw material/mL significantly reversed corticosterone- and glutamate-induced neurotoxicity in a dose-dependent manner. The fractions containing phenolic acid derivatives (0.59% w/w in the flowers) and/or flavonoids (0.60% w/w) exerted similar dose-dependent neuroprotective effect whereas the fractions with non-phenolic compounds exhibited no activity. The presence of phenolic acid derivatives in the corticosterone- and glutamate-treated PC12 cells elevated the DA level in the cell culture medium whereas flavonoids resulted in increased ACH and 5-HT levels. CONCLUSION: Phenolic acid derivatives and flavonoids were likely the active constituents of daylily flowers and they conferred a similar extent of neuroprotection, but affected the release of neurotransmitters in a different manner.