Grifolin Induces Cell Death of Human Lung Cancer A549 Cell Line via Inhibiting KRAS-Mediated Multiple Signaling Pathways.

In the current work, grifolin was obtained from the twigs and leaves of Daphne genkwa for the first time and displayed significant growth inhibition against human lung carcinoma A549 cells. Subsequent in vitro antitumor evaluation revealed that grifolin could induce remarkable cell apoptosis and G0/G1 phase arrest, as well as block cell migration and invasion. In addition, grifolin also disrupted cellular energy metabolism by inducing reactive oxygen species, reducing adenosine triphosphate and mitochondrial membrane potential, and damaging DNA synthesis. Further RNA-seq analysis demonstrated that treatment of grifolin on A549 cells led to gene enrichment in MAPK, PI3K/Akt and NF-κB signaling pathways, all of which were inhibited by grifolin according to immunoblotting experiments. Further mechanistical studies disclosed that the expression of a key upstream protein KRAS was also blocked, and the cell death triggered by grifolin could be rescued by a RAS activator ML-099. Moreover, pretreatment of ML-099 on A549 cells could reverse the grifolin-induced downregulation of key proteins in the three aforementioned pathways. These findings indicate that grifolin could induce cell death in A549 cell line by inhibiting KRAS-mediated multiple signaling pathways.

Target isolation of diverse sesquiterpenoid from the stems of Daphne genkwa based on molecular networking.

Guiding by LC-MS/MS analysis and the Global Natural Products Social (GNPS) Molecular Networking, three undescribed sesquiterpenoids, stedapgens A-C, and two known analogues were discovered in the barks of Daphne genkwa Sieb. et Zucc. The structures were determined by analysis of their spectroscopic data and quantum-chemical calculations. All the isolated novel compounds were tested for their acetylcholinesterase inhibitory activities with IC50 = 0.754 ± 0.059, 0.696 ± 0.026, and 0.337 ± 0.023 µg/ml. Among them, stedapgen A displayed promising inhibitory activities against AChE, and the binding sites were predicted by molecular docking.

Meroterpenoids from Daphne genkwa shows promising in vitro antitumor activity via inhibiting PI3K/Akt/mTOR signaling pathway in A549 cells.

Phytochemical investigation into the leaves and branches of Daphne genkwa afforded 25 meroterpenoids (1-16) including nine pairs of enantiomers (1a/1b-8a/8b and 12a/12b), among which 20 compounds have been reported in the present work for the first time. The structures with absolute configurations of the new molecules (excluding 10-13) were established via comprehensive spectroscopic analyses especially electronic circular dichroism (ECD) and Mosher's methods. A preliminary in vitro cell viability assay revealed remarkable cytotoxicities of selective compounds against A549 (lung), Hela (cervical), MDA-MB231 (breast) and MCF-7 (breast) cancer cells, and compound 8a showed the best inhibitory activity with IC50 values in the range of 3.12-4.67 µM toward the four cell lines. Subsequent in vitro antitumor evaluation of 8a disclosed that it could inhibit the proliferation and metastasis, as well as induce significant apoptosis and cycle arrest, of A549 cells. Further mechanistic investigations revealed that 8a could exert its antitumor activity via inhibiting the PI3K/Akt/mTOR signaling pathway.

Identification of Daphne genkwa and Its Vinegar-Processed Products by Ultraperformance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry and Chemometrics.

Crude herbs of Daphne genkwa (CHDG) are often used in traditional Chinese medicine to treat scabies baldness, carbuncles, and chilblain owing to their significant purgation and curative effects. The most common technique for processing DG involves the use of vinegar to reduce the toxicity of CHDG and enhance its clinical efficacy. Vinegar-processed DG (VPDG) is used as an internal medicine to treat chest and abdominal water accumulation, phlegm accumulation, asthma, and constipation, among other diseases. In this study, the changes in the chemical composition of CHDG after vinegar processing and the inner components of the changed curative effects were elucidated using optimized ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Untargeted metabolomics, based on multivariate statistical analyses, was also used to profile differences between CHDG and VPDG. Eight marker compounds were identified using orthogonal partial least-squares discrimination analysis, which indicated significant differences between CHDG and VPDG. The concentrations of apigenin-7-O-ß-d-methylglucuronate and hydroxygenkwanin were considerably higher in VPDG than those in CHDG, whereas the amounts of caffeic acid, quercetin, tiliroside, naringenin, genkwanines O, and orthobenzoate 2 were significantly lower. The obtained results can indicate the transformation mechanisms of certain changed compounds. To the best of our knowledge, this study is the first to employ mass spectrometry to detect the marker components of CHDG and VPDG.

Effects of the total flavonoid extracts and the monomers of Daphne genkwa on CYP2C8 activity.

This study aimed to assess the effects of total flavonoid extracts (TFDG) and the monomers of Daphne genkwa on the CYP2C8 activity in vitro and in vivo.The 50% inhibitory concentration (IC50) values were used to determine the inhibitory effect of TFDG and its four monomers for the CYP2C8 activity by recombinant human CYP2C8 (RHCYP2C8) yeast microsome system in vitro, and the volume per dose index (VDI) was predicted the potential inhibition in vivo. The effects of multiple-dose administration of TFDG on the pharmacokinetic parameters of rosiglitazone in rats were evaluated.The IC50 values of apigenin, luteolin, hydroxy-genkwanin, genkwanin, and TFDG were 7.27 µmol/L, 11.9 µmol/L, 28.1 µmol/L, 127 µmol/L, and 13.4 µg/mL, respectively. The VDI values of apigenin and TFDG were 2.15 L and 6.60 L. In vivo study, compared with the control group, the elimination phase half-life and mean residence time in the TFDG treatment group were significantly increased by 96.9% and 106.8% (p <.05), respectively.Apigenin showed a moderate inhibitory effect on the CYP2C8 activity in vitro, while the other three monomers were weak inhibitors. TFDG had a strong inhibitory effect on CYP2C8 in vitro and in vivo, and also inhibited the metabolism of rosiglitazone in rats.

Discovery of guaiane-type sesquiterpenoids from the roots of Daphne genkwa with neuroprotective effects.

A phytochemical study on the roots of Daphne genkwa yielded seven new guaiane-type sesquiterpenoids (1-7), daphne A-G. Their structures were elucidated through comprehensive spectroscopic analyses. The absolute configurations were determined by comparison between experimental electronic circular dichroism (ECD) and calculated ECD spectra via time-dependent density functional theory (TDDFT) and the modified Mosher's method. Furthermore, all isolates were evaluated for their neuroprotective activities against H2O2-induced injury in human neuroblastoma SH-SY5Y cells. Among them, compounds 1 (78.42%) and 4 (79.34%) exhibited potent neuroprotective effects against H2O2-induced neurotoxicity at 25 µM. Further Annexin V-FITC/propidium iodide (PI) doubling staining exhibited that the neuroprotective effects of compounds 1 and 4 appeared to be mediated via suppressing cell apoptosis. Flow cytometry assays also proved that compounds 1 and 4 could attenuate mitochondrial dysfunction in SH-SY5Y cells.

Hydroxygenkwanin Suppresses Non-Small Cell Lung Cancer Progression by Enhancing EGFR Degradation.

Epidermal growth factor receptor (EGFR) is frequently overexpressed and mutated in non-small cell lung cancer (NSCLC), which is the major type of lung cancer. The EGFR tyrosine kinase inhibitors (TKIs) are the approved treatment for patients harboring activating mutations in the EGFR kinase. However, most of the patients treated with EGFR-TKIs developed resistance. Therefore, the development of compounds exhibiting unique antitumor activities might help to improve the management of NSCLC patients. The total flavonoids from Daphne genkwa Sieb. et Zucc. have been shown to contain antitumor activity. Here, we have isolated a novel flavonoid hydroxygenkwanin (HGK) that displays selective cytotoxic effects on all of the NSCLC cells tested. In this study, we employed NSCLC cells harboring EGFR mutations and xenograft mouse model to examine the antitumor activity of HGK on TKI-resistant NSCLC cells. The results showed that HGK suppressed cancer cell viability both in vitro and in vivo. Whole-transcriptome analysis suggests that EGFR is a potential upstream regulator that is involved in the gene expression changes affected by HGK. In support of this analysis, we presented evidence that HGK reduced the level of EGFR and inhibited several EGFR-downstream signalings. These results suggest that the antitumor activity of HGK against TKI-resistant NSCLC cells acts by enhancing the degradation of EGFR.

A new flavanol from the roots of Daphne genkwa.

The phytochemical investigation of the roots of Daphne genkwa yielded six secondary metabolites, including a new flavanol derivative, (2R, 3S)-5,7,4'-trihydroxy-8-methoxycarbonylflavanol (1), and five known compounds (2-6). The molecular structures of the isolated constituents were elucidated on the basis of extensive spectroscopic analysis, including UV, IR, NMR, and MS, and comparison with literature data. Furthermore, the cytotoxic activity of 1 and 2 against A549, HL-60, SMMC-7721, MCF-7, and SW480 cell lines was also described.

Simultaneous determination of three flavonoids and one coumarin by LC-MS/MS: Application to a comparative pharmacokinetic study in normal and arthritic rats after oral administration of Daphne genkwa extract.

A selective and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for investigating the pharmacokinetics of umbelliferone, apigenin, genkwanin and hydroxygenkwanin after oral administration of Daphne genkwa extract. Plasma samples were treated by protein precipitation with acetonitrile. Analytes were detected by triple-quadrupole MS/MS with an ESI source in negative selection reaction monitoring mode. The transitions of m/z 161 → 133 for umbelliferone, m/z 269 → 117 for apigenin, m/z 283 → 268 for genkwanin and m/z 299 → 284 for hydroxygenkwanin were confirmed for quantification. Chromatographic separation was conducted using an Eclipse XDB-C18 column, and the applied isocratic elution program allowed for simultaneous determination of the four analytes for a total run time of 2.5 min. The linearity was validated over the plasma concentration ranges of 1.421-1421 ng/mL for umbelliferone, 0.845-845 ng/mL for apigenin, 1.025-1025 ng/mL for genkwanin and 0.845-845 ng/mL for hydroxygenkwanin. The extraction recovery rate was >82.7% for each analyte. No apparent matrix effect was observed during the bioanalysis. After full validation, the proposed method was successfully applied to compare the pharmacokinetics of these analytes between normal and arthritic rats.

[Vinegar processing attenuates toxicity on IEC-6 cells caused by chloroform extraction of Daphne genkwa].

To screen the toxic polar fractions of Daphne genkwa, compare the toxicity of D. genkwa on crypts epithelial cells IEC-6 before and after vinegar processing, and preliminarily investigate the mechanism of D. genkwa vinegar processing on toxicity reducing. The proliferation of IEC-6 cells was observed by MTT. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), lactate dehydrogenase (LDH), as well as the enzyme activity of Na⁺-K⁺-ATPase and Ca²âº-Mg²âº-ATPase were determined in IEC-6 cells to evaluate the oxidative damages degree of IEC-6 cells. The apoptosis and cell cycle were analyzed by Flow Cytometry. The results showed that the dichloromethane extraction was the toxic polar fraction of D. genkwa, and after vinegar processing, the toxicity of dichloromethane fraction was significantly reduced (P<0.01). As compared with the blank control group, the dichloromethane fraction of D. genkwa can obviously decrease the levels of SOD, Na⁺-K⁺-ATPase, Ca²âº-Mg²âº-ATPase (P<0.01) and content of GSH, but increase the level of LDH and MDA in cell supernatant (P<0.01). Besides, it obviously increased the early and late apoptotic rate of IEC-6 cells, obviously decreased the proportion of G1stage cells, increased the ratio of S stage cells and M stage cells (P<0.01). After vinegar processing, as compared with D. genkwa groups of various doses, it can significantly increase the levels of SOD, Na⁺-K⁺-ATPase, Ca²âº-Mg²âº-ATPase (P<0.01) and content of GSH, decrease the level of LDH, MDA(P<0.01), significantly decrease the early and late apoptosis rate of IEC-6 cells (P<0.01), increase the proportion of G1stage cells, and decrease the ratio of S stage cells and M stage cells (P<0.01). Vinegar processing can reduce the toxicity of dichloromethane fraction of D. genkwa, and its mechanism may be associated with improving the activity of antioxidant enzymes and permeability in IEC-6 cells, and decreasing the oxidative damage.

Daphnane and Phorbol Diterpenes, Anti-neuroinflammatory Compounds with Nurr1 Activation from the Roots and Stems of Daphne genkwa.

The methanol extract of the roots and stems of Daphne genkwa and its constituents yuanhuacin (1) and genkwanine N were previously reported to have Nurr1 activating effects and neuroprotective effects in an animal model of Parkinson's disease (PD). In this study, four more daphnane-type diterpenes (acutilonine F (2), wikstroemia factor M1 (3), yuanhuadine (5), and yuanhuatine (6)) and two phorbol-type diterpenes (prostratin Q (4) and 12-O-n-deca-2,4,6-trienoyl-phorbol-(13)-acetate (7)) were isolated as Nurr1 activating compounds from the D. genkwa extract. Consistent with their higher Nurr1 activating activity, compounds 1, 4, 5, and 7 exhibited higher inhibitory activity on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in murine microglial BV-2 cells with an IC50 (µM) of 1-2, which was 15-30 times more potent than that of minocycline (29.9 µM), a well-known anti-neuroinflammatory agent. Additionally, these diterpenes reduced expression and transcription of LPS-induced pro-inflammatory cytokines in BV-2 cells. Thus, the daphnane-type and phorbol-type diterpenes had anti-neuroinflammatory activity with Nurr1 activation and could be responsible for the anti-PD effect of the roots and stems of D. genkwa.

Relationship between the UPLC-Q-TOF-MS fingerprinted constituents from Daphne genkwa and their anti-inflammatory, anti-oxidant activities.

Daphne genkwa Sieb.et Zucc. is a well-known medicinal plant. This study was designed to apply the ultra-high performance liquid chromatography system to establish a quality control method for D. genkwa. Data revealed that there were 15 common peaks in 10 batches of D. genkwa Sieb. Et Zucc. (Thymelaeaceae) from different provinces of China. On this basis, the fingerprint chromatogram was established to provide references for quality control. Afterwards, the chemical constitutions of these common peaks were analyzed using the UPLC-Q-TOF-MS system and nine of them were identified. In addition, LPS-stimulated RAW264.7 murine macrophages and DPPH assay were used to study the anti-inflammatory and anti-oxidation effects of D. genkwa. Then the fingerprint-efficacy relationships between UPLC fingerprints and pharmacodynamic data were studied with canonical correlation analysis. Analysis results indicated that the anti-inflammatory and anti-oxidation effects differed among the 10 D. genkwa samples owing to their inherent differences of chemical compositions. Taken together, this research established a fingerprint-efficacy relationship model of D. genkwa plant by combining the UPLC analytic technique and pharmacological research, which provided references for the detection of the principal components of traditional Chinese medicine on bioactivity.

[Morphological and microscopical identification of Genkwa Ramulus and its adulterants].

The purpose of this article is to identify Daphne genkwa and its adulterants, Wikstroemia chamaedaphne, according to the morphological and microstructure characteristics of their stem and foliage. The root of D.genkwa was studied simultaneously. The results indicated that the crude drug and processed pieces of Genkwa Ramulus were mainly composed of stems and branches where obvious opposite petiole scars and branch marks were able to be seen on their nodes. Otherwise, foliage or peduncles generally couldn't be found. Moreover, the fine silver flocculent fibers could be observed in the bark of fracture surface. The adulterants were the plant segments which were composed of stems, foliage and peduncles with spikelet-pedicel scars. There existed microstructures differences between Genkwa Ramulus and its adulterants. In the former, single thick lignified phloem fibers were interspersed in the stem phloem of the transverse section with very thick wall and unicellular non-glandular hairs could be observed on the lower epidermis of foliage. Nevertheless, in the latter, there was no thick lignified phloem fibers in cross section of stem phloem, the outer wall of epidermal cells of foliage hadthick cuticles and no non-glandular hairs in lower epidermis of foliage. The results can be used for the identification and the quality standard of the crude drug and processed pieces of D.genkwa.The characteristics of the microstructures and the transverse section can be used to identify the radix D.genkwa.

Antitumor Activity of Total Flavonoids from Daphne genkwa in Colorectal Cancer.

Daphne genkwa Sieb.et Zucc. is a well-known medicinal plant. This study was designed to investigate the anticancer effects of total flavonoids in D. genkwa (TFDG) in vitro and in vivo. HT-29 and SW-480 human colorectal cancer cells were cultured to investigate the anticancer activity of TFDG. In addition, the Apc(Min/+) mouse model was applied in the in vivo experiment. Results of the cell experiment revealed that TFDG possessed significant inhibitory effects on HT-29 and SW-480 human colorectal cancer cells (both p < 0.01). Furthermore, our in vivo data showed that after treatment with TFDG, there was a significant increase in life span (both p < 0.01) and tumor numbers were reduced in the colon (both p < 0.01), which was supported by the data of tumor distribution, body weight changes and organ index. Our results also indicated that expressions of interleukin (IL)-1α, IL-1ß, IL-6, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in gut tissue were downregulated by treatments of TFDG, and immunity cytokine secretions in the serum were regulated after oral administration of TFDG. Taken together, these findings suggested that TFDG has a potential clinical utility in colorectal cancer therapeutics, and TFDG's action is likely linked to its ability to regulate immune function and inhibit the production of inflammatory cytokines.

Daphne genkwa: Ethnopharmacology, phytochemistry and pharmacology of an important traditional Chinese medicine.

Daphne genkwa, as a traditional medicine, is widely distributed in China, Korea and Vietnam. In China, the dried flower buds of this plant are named "Yuanhua". It has the ability to effectively promote urination, eliminate phlegm and alleviate cough, eliminate parasites and cure of scabies, with a broad spectrum of pharmacological effects and considerable clinical efficacy. This paper provides a summary and classification of the main chemical constituents of D. genkwa based on a review of relevant domestic and foreign literature. It also outlines the current research status of traditional clinical usage, pharmacological effects, and toxicity of D. genkwa. The aim is to provide a theoretical basis for further study of D. genkwa and its potential new clinical applications.

Bioactive daphnane diterpenes from the flower buds of Daphne genkwa (Thymelaeaceae).

Four diterpenes of the daphnane type were isolated from a methanol extract of the flower buds of Daphne genkwa, the two of them were new structures named genkwadanes J (1) and K (2). Their structures were determined based on analysis of their 1D- and 2D-NMR, HRESIMS and ECD calculations. Among the isolates, the cytotoxicity was assessed via the MTT method using the K562, MCF-7 and HeLa cancer cell lines, the positive control was taxol. Compounds 1 and 3 exhibited appreciable cytotoxic activity against the K562 cancer cell line with IC50 values between 6.58 and 5.33 µM. Compounds 2 and 4 showed noteworthy inhibitory effects against the MCF-7 cell line with IC50 values of 3.25 and 2.56 µM, respectively. All compounds showed weak cytotoxicities to the Hela cell line with IC50 values in the range of 20.19-55.23 µM.

Ultrasound-assisted extraction, optimization, and purification of total flavonoids from Daphnegenkwa and analysis of their antioxidant, anti-inflammatory, and analgesic activities.

Daphne genkwa (D. genkwa) is the dried flower buds of a Chinese medicinal plant with multiple biological activities. Response surface methodology (RSM) combined with artificial neural network (ANN) techniques were utilized to optimize ultrasound-assisted extraction conditions for D. genkwa. Antioxidant activity and anti-inflammatory and analgesic properties of total flavonoids from D. genkwa (TFDG) were assessed. Optimal conditions involving ultrasonic power of 225 W, 30 min extraction time, 30 mL/g liquid-solid ratio, 60 °C extraction temperature, and 70% ethanol concentration yielded a maximum total flavonoids content (TFC) of 5.41 mg/g. After microporous resin purification, four specific flavonoids in D. genkwa were identified and quantified using high-performance liquid chromatography (HPLC). The TFDG demonstrated potent antioxidant activity, with a 94% rate of scavenging the 2, 2-diphenyl-1-picrylhydrazyl (DPPH). Furthermore, TFDG exhibited pain-alleviating properties in hot plate and acetic acid-induced writhing tests and noteworthy inhibitory effects on xylene-induced ear swelling in mice. The total flavonoids extracted by ultrasound had excellent biological activity. This establishes a foundation for further investigation into the potential medical value of D. genkwa.

Daphne genkwa flower extract promotes the neuroprotective effects of microglia.

BACKGROUND: Microglia are innate immune cells in the central nervous system that play a crucial role in neuroprotection by releasing neurotrophic factors, removing pathogens through phagocytosis, and regulating brain homeostasis. The constituents extracted from the roots and stems of the Daphne genkwa plant have shown neuroprotective effects in an animal model of Parkinson's disease. However, the effect of Daphne genkwa plant extract on microglia has yet to be demonstrated. PURPOSE: To study the anti-inflammatory and neuroprotective effects of Daphne genkwa flower extract (GFE) in microglia and explore the underlying mechanisms. METHODS: In-vitro mRNA expression levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), inducible nitric oxide synthase, Arginase1, and brain derived neurotropic factor (BDNF) were analyzed by reverse transcription polymerase chain reaction in microglia cells. Nitric oxide (NO) and TNF-α protein were respectively analyzed by Griess reagent and Enzyme Linked Immunosorbent Assay. Immunoreactivity of Iba-1, Neu-N, and BDNF in mouse brain were analyzed by immunofluorescence staining. Phagocytosis capacity of microglia was examined using fluorescent zymosan-red particles. RESULTS: GFE significantly inhibited lipopolysaccharide (LPS)-induced neuroinflammation and promoted neuroprotection both in vitro and in vivo. First, GFE inhibited the LPS-induced inflammatory factors NO, iNOS, and TNF-α in microglial cell lines and primary glial cells, thus demonstrating anti-inflammatory effects. Arginase1 and BDNF mRNA levels were increased in primary glial cells treated with GFE. Phagocytosis was also increased in microglia treated with GFE, suggesting a neuroprotective effect of GFE. In vivo, neuroprotective and anti-neuroinflammatory effects of GFE were also found in the mouse brain, as oral administration of GFE significantly inhibited LPS-induced neuronal loss and inflammatory activation of microglia. CONCLUSION: GFE has anti-inflammatory effects and promotes microglial neuroprotective effects. GFE inhibited the pro-inflammatory mediators and enhanced neuroprotective microglia activity by increasing BDNF expression and phagocytosis. These novel findings of the GFE effect on microglia show an innovative approach that can potentially promote neuroprotection for the prevention of neurodegenerative diseases.

Tigliane-and daphnane-type diterpenoids from the buds of Daphne genkwa with their cytotoxic activities.

Two new tigliane- and daphnane-type diterpenoids, given the trivial names daphnegens A-B (1-2) were isolated from the buds of Daphne genkwa. Their structures were assigned on the basis of extensive spectroscopic. The absolute configurations of both compounds were determined by comparison of their calculated and experimental CD curves. In addition, compounds 1-2 were tested for their cytotoxic activities against MCF-7 and HepG-2 human cancer cell lines, and compound 2 showed remarkable cytotoxic activity against HepG-2 cell line with the IC50 value of 11.5 µM.

Design of Experiments-Based Optimization of Flavonoids Extraction from Daphne genkwa Flower Buds and Flavonoids Contents at Different Blooming Stages.

The flower buds of Daphne genkwa have been reported as a potent resource associated with anti-angiogenic, anti-tumor, anti-rheumatoid arthritis activities, as well as immunoregulation. This paper aimed to establish an optimal extraction method for flavonoids, as active phytochemicals, and to conduct a comparative analysis by profiling the different blooming stages. Optimized shaking extraction conditions from the design of experiments (DoE), such as minutely mixture design, 23 full factorial design, and polynomial regression analysis, involved an agitation speed of 150 rpm and temperature of 65 °C for 12 h in 56% (v/v) acetone solvent. After, a comparative analysis was performed on three blooming stages, juvenile bud, mature purple bud, and complete flowering, by ultra-high-performance liquid chromatography-photodiode array-mass spectrometry (UHPLC-PDA-MS). Most flavonoids increased during bud growth and then decreased when the bud opened for blooming. In particular, apigenin 7-O-glucuronide, genkwanin 5-O-primeveroside, and genkwanin strikingly showcased this pattern. Furthermore, the raw spectrometric dataset was subjected to orthogonal projection to latent structures discriminant analysis (OPLS-DA) to find significant differences in the flavonoids from the juvenile bud, mature purple bud, and complete flowering. In conclusion, the present study facilitates an understanding of flavonoid change at different blooming stages and provides a momentous reference in the research of D. genkwa.