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| ORIGINAL ARTICLE |
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| Year : 2023 | Volume
: 66
| Issue : 5 | Page : 313-325 |
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Daylily (Hemerocallis fulva Linn.) flowers improve sleep quality in human and reduce nitric oxide and interleukin-6 production in macrophages
Li-Min Hsu1, Hua-Wei Chen2, Po-Ching Wu3, Kuo-Feng Hua4
1 Department of Biotechnology and Animal Science, National Ilan University; Department of Nursing, St. Mary's Junior College of Medicine, Nursing and Management, Yilan, Taiwan
2 Department of Chemical and Materials Engineering, National Ilan University, Yilan, Taiwan
3 Department of Biomechatronic Engineering, National Ilan University, Yilan, Taiwan
4 Department of Biotechnology and Animal Science, National Ilan University, Yilan; Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, Taiwan
| Date of Submission | 14-Mar-2023 |
| Date of Decision | 31-May-2023 |
| Date of Acceptance | 06-Jun-2023 |
| Date of Web Publication | 15-Sep-2023 |
Correspondence Address:
Prof. Kuo-Feng Hua
No. 1, Sec. 1, Shennong Rd., Yilan City, Yilan County 260
Taiwan
Prof. Po-Ching Wu
No. 1, Sec. 1, Shennong Rd., Yilan City, Yilan County 260
Taiwan
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/cjop.CJOP-D-23-00043
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.
Keywords: Anti-inflammation, daylily flowers, insomnia, sleep quality
How to cite this article:
Hsu LM, Chen HW, Wu PC, Hua KF. Daylily (Hemerocallis fulva Linn.) flowers improve sleep quality in human and reduce nitric oxide and interleukin-6 production in macrophages. Chin J Physiol 2023;66:313-25 |
How to cite this URL:
Hsu LM, Chen HW, Wu PC, Hua KF. Daylily (Hemerocallis fulva Linn.) flowers improve sleep quality in human and reduce nitric oxide and interleukin-6 production in macrophages. Chin J Physiol [serial online] 2023 [cited 2023 Nov 30];66:313-25. Available from: https://www.cjphysiology.org/text.asp?2023/66/5/313/385877 |
| Introduction | |  |
Insomnia is the most common sleep complaint in older adults.[1] Approximately one-third of the population worldwide experiences insomnia accompanied by daytime dysfunction.[2] Self-reports of sleep onset latency (SOL), wake time after sleep onset (WASO), or total sleep time (TST) surpass predetermined thresholds for insomnia (e.g. SOL or WASO >30 min; TST <6 h).[3] Increased studies support a bidirectional relationship between sleep disturbance and inflammation.[4],[5],[6],[7] Good sleep quality enhances immunity and that afferent signals from immune cells promote sleep.[8] Otherwise, inflammation and oxidative stress has been implicated in sleep disturbance-related disorders.[9] Mild insomnia is frequently self-treated with over-the-counter medications, herbs, or non-medicinal strategies, such as sleep hygiene, cognitive behavioral therapy, and sleep restriction therapy to modify precipitating or contributory factors.[10] However, the limited use of pharmacological treatments is due to the undesirable side effects, such as performance and memory impairment, residual sedation, falls, undesired behaviors during sleep, somatic symptoms, and drug interactions.[11] There is a need to develop nutraceutical strategy to help improve sleep quality.[12]
Daylily (Hemerocallis fulva) flowers, both fresh and dried, have been used as a vegetable and medicinal herb for 1000 of years in Taiwan and eastern Asia. Flavonoids-containing extract of H. fulva leaves had a strong radical cation ABTS+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and OH radical scavenging activity and reduced reactive oxygen species (ROS) production in H2O2-stimulated human immortal keratinocyte HaCaT cells.[13] Aqueous and ethanol extract H. fulva reduced the expression levels of ROS and pro-inflammatory mediators, and increased the expression levels of anti-oxidative enzymes in high glucose-stimulated human umbilical vein endothelial cells.[14] Caffeoylquinic acids isolated from the aqueous ethanol extract of H. fulva flowers reduced tumor necrosis factor-α (TNF-α)-induced ROS production in human hepatoma HepG2 cells.[15] The antioxidant activity of ethanol extract of H. fulva flowers was confirmed in vivo, as increased superoxide dismutase activity and reduced lipid peroxidation was found in both blood and liver of H. fulva flowers-feeding rat.[16] The aqueous methanol extract of fresh H. fulva leaves also exerted antioxidant activity by inhibiting lipid oxidation.[17] In addition, two new phenanthrenes, Hemecitones A and B, isolated from H. fulva showed inhibitory effect against breast, hepatocellular, and lung carcinoma cell lines in vitro.[18] The compounds in H. fulva flowers were predicted to against depressive disorder by a combined network pharmacology and molecular docking approach.[19] Increasing evidences show that phytochemicals with anti-oxidative and anti-inflammatory activity have potential to improve sleep quality.[20] The slow wave sleep and paradoxical sleep of mice receiving freeze-dried flowers of the H. fulva were increased during the dark period compared to the control mice.[21] However, the effects of H. fulva flowers on sleep quality in human and inflammatory response in macrophages have not been investigated.
Insomnia is commonly associated with inflammation and oxidative stress, in particularly, with higher blood interleukin-6 (IL-6) level of insomnia patients.[9],[22] The importance of natural products with anti-inflammatory and anti-oxidative activities has attracted the interest of researchers, as they have potential to improve the sleep quality.[20] Rutin is a low molecular weight flavonoid glycoside with potent anti-oxidative activity.[23] Rutin reduced lipopolysaccharide (LPS)-induced acute cardiac and lung injuries in mice by decreasing the production of nitric oxide (NO) and IL-6, respectively.[24],[25] Developing effective dietary supplements or nutraceutical as sleep aids with few side effects has attracted the attention of researchers and biotech and food industries.[12] In this study, we investigate whether H. fulva Linn. flowers can improve sleep quality in free-living adults with sleep disorders using subjective sleep questionnaires and objective sleep measurements, including heart rate variability (HRV) and Xiaomi Mi Smart Band 3 sleep monitoring. We also investigate the anti-inflammatory potential of H. fulva Linn. flowers in macrophages and the rutin contents of H. fulva flowers extracts.
| Materials and Methods | | |
Materials
LPS from Escherichia coli 0111:B4 and chemicals analytical grade solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from MD Bio, Inc. (Taipei, Taiwan). Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Isand, NY, USA). The H. fulva Linn. flowers (Taitung No. 6) were purchased from Taitung, Taiwan. IL-6 and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (Minneapolis, MN, USA).
Preparation of daylily flowers samples
The H. fulva Linn. flowers were confirmed for sulfur-, heavy metal- and pesticide residues-free. The daylily flowers were air-dried, pulverized and passed through a sieve to give a homogeneous powder. The powder was maintained at −20°C before use. For human clinical trial, one tea bag contained 3 g of daylily flowers powder. For daylily flowers extraction, the powder (150 g) was extracted with 1.5 L of ddH2O at 4°C for 48 h (stir speed: 120 rpm). The soluble extract was filtered and dried to give an aqueous extract, named the daylily flowers aqueous extract (DFAE). The residues were then extracted with 1.5 L of 95% ethanol at 4°C for 48 h (stir speed: 120 rpm). The extracts were evaporated under reduced pressure in a rotary vacuum evaporator to give an ethanolic extract, named the daylily flowers ethanolic extract (DFEE). To yield soluble fractions of ethyl acetate (EtOAc) and water (ddH2O), DFEE was successively liquid-liquid partitioned with EtOAc and ddH2O (each volume was 250 mL and was repeated six times). The soluble fractions of EtOAc and water were dried, named the EtOAc-soluble fraction (EF) and water-soluble fraction (WF). The EF was then subjected to silica gel column chromatography with successive elution by a hexane/EtOAc gradient solvent system to obtain six fractions (EF-1~EF-6). All of the sample preparation procedures are shown in [Figure 1].
Participants
The randomized clinical trial was conducted at the National Ilan University and St. Mary's Junior College of Medicine, Nursing and Management between September 2019 and August 2021. The study protocol was approved by the Institutional Review Board of National Yang-Ming University Hospital (Clinical Trial Registration Number: 2019B004). Participants were free-living adults between 20 and 60 years of age with a self-diagnose of insomnia. Inclusion criteria included adults who did not consume alcohol, limited caffeinated beverages to one cup or can a day consumed before noon, and worked and rested at the recommended times. Every participant signed an informed consent form before the program started. Participants were screened using the previously validated Chinese version of the Pittsburgh Sleep Quality Index (CPSQI). A global CPSQI score ≥ 5 was used as the cut-off value indicating poor sleep.[26] Exclusion criteria included suffering from physical disorders that could affect night sleep, such as angina pectoris and kidney dialysis; having sleep abnormalities, including arrhythmia, intermittent limb movement abnormalities, and sleep-related breathing disorders; being pregnant (or having the possibility of having children) or breastfeeding; and working as shift workers. This study is a randomized clinical trial in which participants were assigned to one of two groups [Figure 2]. Ten participants self-reported their sleep quality as very poor, and the remaining 26 self-reported poor sleep quality.
Study design of randomized clinical trial
Participants in this study were randomly divided into two groups of equal size to evaluate their physical state, questionnaires, and basic demographic characteristics and to explore the effect of the experimental group's intervention. The experimental group was administered a drink consisting of 150–200 mL of hot water extract of daylily flowers powder (3 g/pack) 30 min before going to bed for 4 weeks. The hot water extract was made by brewing the pack of daylily flowers powder with hot water (90–100°C) for 25–30 min. The control group drank water. All participants were briefly introduced to the study by the Co-Principal Investigator to enter the follow-up evaluation phase. Participants completed the basic demographic sheet and CPSQI; once the CPSQI score confirmed that they had poor sleep quality, they were enrolled in the formal study. Heart rate is modulated by the combined effects of the sympathetic and parasympathetic nervous systems. The changes in heart rate over time (HRV) provide information about autonomic functioning. HRV has been applied to understand autonomic changes during different sleep stages.[27] HRV has been applied to analyze the effect of sleep-disordered breathing, periodic limb movements and insomnia both during sleep and during the daytime.[28],[29],[30] On weeks 0 and 4, participants filled out the CPSQI questionnaire, and their HRV was measured; the Xiaomi Mi Smart Band 3 was worn by all participants every night during the study duration [Figure 3].
Study instruments
The CPSQI comprises questions that assess subjects' sleep quality and sleep disturbance for 1 month. The CPSQI includes seven items: sleep quality, sleep latency, sleep duration, sleep efficiency, sleep disturbance, use of sleeping medications, and daytime functioning. Each item is scored from 0 to 3, and the total score is 21 points. The higher the score, the worse quality of sleep, with a cut off score of 5.[31] The following data were collected using Xiaomi Mi Smart Band 3: (1) time to fall asleep; (2) time to wake up; (3) how much are you awake during sleep; (4) how much total sleep; (5) how much in deep sleep; and (6) how much in light sleep. The Xiaomi Mi Smart Band 3 requires certain personal data from the user to precisely calculate the activity, such as age, height, weight, gender, handedness, and wristband location. Wristband data will be obtained from the Mi Fit application, which is the native app of the Xiaomi Mi Band 3 wristband. It is necessary to connect the wristband via Bluetooth to a smartphone of general use for the group, creating generic E-mails for each of the wristbands.[32] Polysomnography (PSG) is considered by the scientific community as the most reliable test for the measurement of sleep parameters.[33] The level of agreement between both devices for light sleep identification (the proportion of PSG-classified N1 + N2 stages identified as light sleep by the Xiaomi wristband), deep sleep identification (the proportion of PSG N3 + N4 stages identified as deep sleep by the Xiaomi wristband).[34] PSG has some clear drawbacks, such as its costs, invasiveness, and time needed for its use.[35],[36] The Xiaomi Mi Band is lower in price, does not interfere with the subjects' night sleep, and has a higher user acceptance,[37] among other advantages. It can provide continuous sleep data for 1 week to monitor sleep patterns among free-living adults who experience sleep disturbances.[34] HRV is a noninvasive method that analyses patterns of consecutive R-peaks in the cardiac cycle for a period of time and is used to assess autonomic nervous system activity. HRV measurement is mainly done with electrocardiography signal analysis. High frequency (HF) % (HF, 0.15–0.5 Hz) is used as an indicator of parasympathetic nerve activity; low frequency (LF) % (LF, 0.05–0.5 Hz) is used as an indicator of sympathetic nerve activity. The LF/HF ratio is used as an indicator of the balance between sympathetic and parasympathetic nerve activity. For this study, NeXus-10 (a 10-channel Bluetooth wireless physiological feedback device) was used for physiological indicators.
Rutin content analysis
The H. fulva Linn. flower extracts DFAE, DFEE, EF, and WF were filtered through a 0.22 μm membrane, and analyzed by high-performance liquid chromatography (HPLC) (Thermo ODS HYPERSIL (C18); 4.6 mm × 150 mm, 5 μm). The mobile phase consisted of acetic acid-water (0.1%) and methanol (99%) using the linear gradient programed. The absorbance was measured at a detection wavelength of 254 nm and the flow-rate was 1 mL/min and the injected volume was 20 μL. The detection limit for rutin was calculated based on a signal-to-noise ratio (S/N) of 0.6 ppm. Five doses of rutin (20, 40, 60, 80, 100 ppm) were injected into HPLC; the linear regression equation for each calibration curve was obtained by plotting the amount of standard compound injected against the peak area. The regression equation and correlation coefficient (R2) were calculated using a Waters 2996 PDA.
Cell culture
Mouse macrophages RAW264.7 and J774A.1 cells were purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan). RAW264.7 and J774A.1 cells were cultured in DMEM medium and RPMI 1640 medium, respectively, containing 10% FBS at 37°C in a 5% CO2 incubator.
Analysis of cell viability
RAW264.7 cells were seeded in 96-well plate at a density of 5 × 105 cells/mL and incubated with vehicle (0.1% DMSO or ddH2O), DFAE, DFEE, EF, WF, or EF-1 to EF-6 for 24 h at the concentration as indicated. The cells were then washed and incubated with 100 μL of MTT reagent (1 mg/mL) at 37°C for 2 h. After MTT reagent was removed, 100 μL of lysis buffer was added to each well and incubation for 30 min. The absorbance was measured at 570 nm using a microplate reader. The cell viability of test samples was determined by the following equation:
(Absorbance of the test group/absorbance of the control group) ×100%.
Analysis of nitric oxide production
RAW264.7 cells were seeded in 24-well plate at a density of 2 × 105 cells in 0.5 mL medium, and incubated with vehicle (0.1% DMSO), DFAE, DFEE or EF-1 to EF-6 for 30 min, followed by stimulated with 100 ng/mL of LPS for additional 24 h. The NO levels in the supernatants were analyzed indirectly by analysis of nitrite levels using the Griess reaction. Briefly, 100 μL of supernatants were mixed with 50 μL of 1% sulfanilamide solution and 50 μL of 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in a 96-well plate for 10 min, and the absorbance was analyzed at 540 nm using a microplate reader.
Analysis of interleukin-6 and tumor necrosis factor-α production
RAW264.7 or J774A.1 cells were seeded in 24-well plate at a density of 2 × 105 cells/mL, and incubated with vehicle (0.1% DMSO), DFAE or DFEE for 30 min, followed by stimulated with 100 ng/mL of LPS for additional 24 h. The IL-6 and TNF-α levels in the supernatants were analyzed by ELISA according to the manufacturer instruction as described previously.[38]
Statistical analysis
Descriptive statistics were used to calculate the distribution of basic demographic data, and all categorical variables are described in count and percentage. Additionally, the Chi-squared test or Fisher's exact test were used depending on the variables analysed. Paired sample t-test was used to analyse the changes of CPSQI, sleep status, and HRV pretest and posttest. The experimental group and the control group were analysed separately. A generalised estimating equation was used to test the effectiveness of the daylily flowers intervention on CPSQI and sleep state. In NO production assay, ANOVA with Dunnett's multiple comparisons test were used for statistical analysis for three or more groups. Error bars represent the standard deviation of three separate experiments. Results with P < 0.05 were considered statistically significant.
| Results | | |
Participants demographics
A total of 36 participants completed the study. The majority of participants were female (77.8%), with most participants between 41 and 50 years old (47.2%), 27.8% self-evaluated with a very poor quality of sleep, and the rest self-evaluated with not good (72.2%). The distribution of the basic variables of the two groups of research subjects was roughly homogeneous (P > 0.05) [Table 1]. | Table 1: Distribution and differences in the demographic characteristics between groups
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Effect of hot water extract of daylily flowers on sleep and heart rate variability parameters
The CPSQI pre-test score was significantly higher than that of the post-test, revealing a significant improvement in CPSQI (P < 0.05) [Table 2]. In addition, the t-test scores for deep sleep (min), deep sleep (%), and light sleep (%) were significant (P < 0.05). When analyzing deep sleep (min) and deep sleep (%), the mean of the post-test was higher than that of the pre-test, indicating significant improvement after the intervention for the experimental group. As for light sleep, the mean of the pretest was higher than that of the posttest, revealing a significant decrease after the intervention [Table 2]. HRV measures improved after the intervention; the t-test score of HF power (HZ) and LF/HF and the mean HF power (HZ) of the post-test was higher than that of the pre-test, indicating an improvement after intervention. The LF/HF mean was higher in the pre-test than in the post-test, indicating a decrease post-intervention. The t-test scores for LF power (HZ), the root mean square of the successive differences (rMSSD) and the standard deviation of the normal to normal inter beat intervals did not reach the significant level (P > 0.05) [Table 2]. The results from sleep parameters and HRV measures showed that the intervention was helpful. Pre-test and post-test t-tests in the control group did not reach a significant level (P > 0.05) for sleep and HRV parameters. However, the control group achieved a significant level in the pre-test and post-test t-tests of the CPSQI (P < 0.05) [Table 3]. | Table 2: The Chinese Version of the Pittsburgh Sleep Quality Index, sleep parameters and including heart rate variability parameters in the experimental group
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 | Table 3: The Chinese Version of the Pittsburgh Sleep Quality Index, sleep parameters and heart rate variability parameters in the control group
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Generalised estimating equation analysis of sleep parameters at different time points
The results of the participants in the control and experimental groups did not reach a significant level in the CPSQI (P > 0.05), indicating no significant difference in the magnitude of changes in the CPSQI between both groups [Table 4]. In addition, the analysis revealed a significant difference in the TST (P = 0.05) and deep sleep (min) (P = 0.02) between control and experimental groups. The changes in TST from the pre-test to the post-test were significantly different between control and experimental groups (P < 0.05), and the degree of increase in the experimental group was higher than that in the control group, showing that the intervention improved TST and deep sleep (min). There was no significant difference between the groups in terms of deep sleep (%), light sleep (min) and light sleep (%) [Table 5]. | Table 4: Generalised estimating equation analysis of Chinese Version of the Pittsburgh Sleep Quality Index at different time points
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 | Table 5: Generalised estimating equation analysis of sleep parameters at different time points
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CPSQI parameters of participants in the experimental group
The CPSQI parameters of experimental group and t-tests of subjective sleep quality, sleep latency, sleep efficiency, and daytime dysfunction reached significant levels (P < 0.05). The post-test mean was lower than the pre-test mean, and the results showed that the intervention was helpful [Table 6]. | Table 6: The Chinese Version of the Pittsburgh Sleep Quality Index parameters in the experimental group
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Effect of daylily flowers extracts on the cell viability of macrophages
Before investigation of the anti-inflammatory potential of daylily flowers in macrophages, we investigate whether the daylily flowers extracts exert potential cytotoxicity. Mouse RAW264.7 macrophages were incubated with daylily flowers extracts for 24 h and the cell viability was analyzed by MTT assay. We found that the cell viability was not affected by DFAE and EF at concentration ≤100 μg/mL, but cell viability was reduced by DFAE [Figure 4]a and EF [Figure 4]b at concentration ≥200 μg/mL. Notably, the cell viability was not affected by DFEE [Figure 4]c and WF [Figure 4]d at concentration ≤400 μg/mL. In addition, the effect of fractions from EF on cell viability was investigated. The result showed that EF-1, EF-2, EF-3 and EF-4 significantly reduced the cell viability at concentration ≥12.5 μg/mL, and EF-5 and EF-6 did not affect the cell viability at concentration ≤100 μg/mL [Figure 4]e. | Figure 4: Effect of daylily flowers extracts on the cell viability of macrophages. RAW264.7 macrophages were incubated with DFAE (a), EF (b), DFEE (c), WF (d) or EF-1 to EF-6 (e) for 24 h, and the cell viability was analyzed by MTT assay. The data are expressed as the mean ± SD of three separate experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to vehicle-treated cells.
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Effect of daylily flowers extracts on nitric oxide production in macrophages
We investigate the NO inhibition potential of daylily flowers extracts on NO production in LPS-activated macrophages. We found that DFAE at concentration ≥50 μg/mL significantly reduced LPS-induced NO production in RAW264.7 macrophages [Figure 5]a, while DFAE at concentration ≥200 μg/mL significantly reduced LPS-induced NO production in J774A.1 macrophages [Figure 5]b. In addition, LPS-induced NO production in RAW264.7 macrophages [Figure 5]c and J774A.1 macrophages [Figure 5]d were significantly reduced by DFEE at concentration ≥ 100 μg/mL and ≥ 200 μg/mL, respectively. We further investigate the NO inhibition potential of EF-1 to EF-6 at concentrations that did not cause significant cell viability loss. We found that EF-1 to EF-4 at concentration ≥ 12.5 μg/mL and EF-5 and EF-6 at concentration of 100 μg/mL significantly reduced NO production in LPS-stimulated RAW264.7 macrophages [Figure 5]e. These results indicate that both aqueous and ethanolic extracts of daylily flowers inhibited NO production in LPS-stimulated macrophages. | Figure 5: Effect of daylily flowers extracts on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated macrophages. RAW264.7 macrophages (a and c) or J774A.1 macrophages (b and d) were incubated with daylily flowers aqueous extract, daylily flowers ethanolic extract or vehicle for 0.5 h, followed by stimulated with LPS for 24 h. The NO levels in the supernatants were analyzed by Griess reaction. (e) RAW264.7 macrophages were incubated with EF-1 to EF-6 or vehicle for 0.5 h, followed by stimulation with LPS for 24 h. The NO levels in the supernatants were analyzed by Griess reaction. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to LPS-treated cells.
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Effect of daylily flowers extracts on interleukin-6 and tumor necrosis factor-α production in macrophages
To further validate the anti-inflammatory effect of daylily flowers extracts, the effect of daylily flowers extracts on IL-6 and TNF-α production in macrophages was studied. We found that DFAE at concentration ≥100 μg/mL significantly reduced LPS-induced IL-6 production in RAW264.7 macrophages [Figure 6]a, while DFAE at concentration ≥50 μg/mL significantly reduced LPS-induced IL-6 production in J774A.1 macrophages [Figure 6]b. DFEE also inhibited IL-6 production in LPS-stimulated RAW264.7 macrophages [Figure 6]c and J774A.1 macrophages [Figure 6]d, while is less potent than DFAE. In addition, DFAE had a tendency to reduce TNF-α production in RAW264.7 macrophages [Figure 7]a and J774A.1 macrophages [Figure 7]b, but not statistically significant. Similar to DFAE, DFEE did not significantly inhibit TNF-α production in RAW264.7 macrophages [Figure 7]c and J774A.1 macrophages [Figure 7]d. These results indicate that both aqueous and ethanolic extracts of daylily flowers inhibited IL-6, but not TNF-α production in LPS-stimulated macrophages. | Figure 6: Effect of daylily flowers extracts on IL-6 production in lipopolysaccharide (LPS)-activated macrophages. RAW264.7 macrophages (a and c) or J774A.1 macrophages (b and d) were incubated with daylily flowers aqueous extract, daylily flowers ethanolic extract or vehicle for 0.5 h, followed by stimulation with LPS for 24 h. The IL-6 levels in the supernatants were analyzed by enzyme-linked immunosorbent assay. **P < 0.01 and ***P < 0.001 compared to LPS-treated cells.
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 | Figure 7: Effect of daylily flowers extracts on tumor necrosis factor-α (TNF-α) production in lipopolysaccharide (LPS)-activated macrophages. RAW264.7 macrophages (a and c) or J774A.1 macrophages (b and d) were incubated with daylily flowers aqueous extract, daylily flowers ethanolic extract or vehicle for 0.5 h, followed by stimulation with LPS for 24 h. The TNF-α levels in the supernatants were analyzed by enzyme-linked immunosorbent assay.
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Rutin content of daylily flowers extracts
To measure the rutin content of daylily flowers, the daylily flower extracts DFAE, DFEE, EF and WF were analyzed by HPLC and the rutin was calculated based on rutin standard. The results showed that daylily flower extracts DFAE, DFEE, EF and WF contain 7.27, 23.30, 14.71 and 57.43 ppm rutin, respectively [Figure 8]. | Figure 8: Rutin content of daylily flowers extracts analyzed by high-performance liquid chromatography.
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| Discussion | | |
This study showed that sleep quality was significantly improved in participants following a 4-week regimen of H. fulva Linn. flowers beverage consumption. The experimental group's post-test subjective questionnaire parameters were significantly lower than those of the pretest, including sleep quality, sleep latency, sleep efficiency, daytime functioning, and CPSQI score. We added the Xiaomi Mi Smart Band 3 as an objective measurement to collect more reliable evidence of sleep quality. After the intervention, the experimental group had a significant increase in the range and ratio of deep sleep. By contrast, the range and ratio of light sleep decreased, indicating a significant improvement in sleep quality. The results obtained from the Xiaomi Mi Smart Band 3 and the subjective questionnaires were consistent.
It has been demonstrated that consumption of freeze-dried flowers of Akinowasuregusa (H. fulva L. var. sempewirona M. Hotta) by C57BL mice significantly increased slow-wave sleep and paradoxical sleep during the dark period (P < 0.05) but did not increase sleep time during the light period. The results showed that the percentage of sleep time within 24 h did not change significantly.[21] Another study showed that H. citrina Baroni effectively regulated the number of night activities and TST and actively regulated sleep attacks and sleep duration in Drosophila melanogaster. The main bioactive components of H. citrina Baroni involved in improving sleep are quercetin, luteolin, kaempferol, caffeic acid and nicotinic acid.[39] In addition, it has been reported that H. citrina flowers and H. fulva flowers have antidepressant effects,[40],[41] with flavonoids as the main active compounds, especially rutin and hesperidin, and high oral doses can cause hypnotic reactions.[42] It has been demonstrated that the body temperature and skin resistance response of patients with insomnia are higher when they are close to falling asleep, and the heartbeat during sleep becomes faster with a reduced heartbeat variability rate.[43] The same group also provided evidence that insomniacs had significantly increased low-frequency power and significantly reduced high-frequency power compared to controls across all sleep stages.[44] Reduced HRV index of parasympathetic tone (normalized HF) in participants with insomnia, who showed increased LF/HF ratios, the standard index of sympathetic nervous system activity, has been reported, compared to the ratios of normal sleepers.[45] Our results showed that the HF in the experimental group increased after intervention (P = 0.88). At the same time, the ratio of LF/HF decreased (P = 0.83), indicating that the participants had parasympathetic nervous system activity during sleep after the intervention. Although our results did not reach a significant different, it showed an improvement trend.
The bidirectional relationship between sleep quality and immune response attracted the attention of scientific community.[4],[5],[6],[7] Sleep disturbances and emotional or physiological stress increase ROS concentration in the body.[46] ROS lead to the endogenous consumption of antioxidants and the subsequent compromise of homeostasis involving neurotransmitter mechanisms.[46] The excessive production of ROS can amplify the inflammatory response, thereby promoting the development of numerous diseases. Consequently, targeting ROS has emerged as a novel approach for both the prevention and treatment of such ailments.[47] Antioxidants have been demonstrated to be potent anti-inflammatory agents that improve sleep quality.[48] Both flowers and leaves of daylily contains phytochemicals that exert anti-oxidative effects in vitro and in vivo,[13],[14],[15],[16],[17] which may explain the benefit effects on improving sleep quality. Among the identified phytochemicals in daylily flowers, rutin is a potent antioxidant that has been reported to improve neurodegenerative disorders.[24] It has been demonstrated that rutin also inhibited NO production in LPS-activated mice and RAW264.7 macrophages.[24],[49] Rutin also reduced IL-6 production in LPS-activated mice.[25] In this study we demonstrated that each daylily flowers H. fulva Linn. extracts contain rutin. The water extract of fresh daylily flower has been reported to inhibit NO production in LPS-activated RAW264.7 macrophages.[50] Same as found in the previous report, we demonstrated that DFAE reduced LPS-induced NO production in both RAW264.7 macrophages and J774A.1 macrophages. However, there is no study investigating the effect of daylily flower ethanolic extract on NO production. Here we found that DFEE and the fractions of EF reduced NO production in LPS-activated RAW264.7 macrophages. DFAE and DFEE both reduced LPS-induced IL-6 production in RAW264.7 macrophages and J774A.1 macrophages. We suggested that the NO and IL-6 inhibitory effects and the sleep quality improvement activity of daylily flower extracts at least in part come from the rutin.
The limitations of this study are the smaller sample size and the total antioxidant capacity or inflammatory indicators were not monitored in the blood of control group and experimental group received daylily flowers. Another limitation is using CPSQI questionnaire to evaluate the quality of specific sleep parameters by subjective measurement. To counteract this limitation, Xiaomi Mi Smart Band 3 was used to monitor sleep objectively and used the NeXus to measure HRV. Although these two devices cannot replace PSG, they can be used as a preliminary assessment tool for users to manage their own sleep quality and consult a health professional when necessary. In addition, these devices can help users to make simple changes of their habits to improve other health problems. Taken together, we found that consumption of daylily flowers can improve the deep sleep range, ratio and sleep efficiency of adults who self-report sleep disorders.
| Conclusion | | |
In this study, we demonstrated that water extract of daylily flowers (H. fulva Linn.) improved sleep quality in people with insomnia. In addition, we also demonstrated that daylily flowers extracts exerted anti-inflammatory effect by reducing the NO and IL-6 production in LPS-activated macrophages. We suggest that daylily flowers have the potential to be a nutraceutical for improving inflammatory-related diseases and sleep quality in the future.
Acknowledgments
This research was funded by Agriculture and Food Agency Council of Agriculture Executive Yuan, R.O.C. (Taiwan) Director Technology Program, grant number 105 Agriculture -3.3.2-Food-Z2, and National Science and Technology Council, Taiwan (MOST 111-2628-B-197-001-MY3).
Financial support and sponsorship
This research was funded by Agriculture and Food Agency Council of Agriculture Executive Yuan, R.O.C. (Taiwan) Director Technology Program, grant number 105 Agriculture -3.3.2-Food-Z2, and National Science and Technology Council, Taiwan (MOST 111-2628-B-197-001-MY3).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]
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