Natural flavonoid; CYP1A enzyme
TGF-β signaling pathway and p53 protein (50 μM Diosmetin upregulates p53 protein expression by ~2.5-fold in HepG2 cells) [1]
- Nuclear Factor-κB (NF-κB) pathway (40 mg/kg Diosmetin reduces NF-κB p65 nuclear translocation by ~65% in mouse pancreatic tissues) [2]

HepG2 cell growth is inhibited by diosmetin in a concentration-dependent manner. HepG2 cells treated with diosmetin are distorted and some take on a round, floating appearance, whereas untreated cells develop normally and have a normal skeleton [1].

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Diosmetin (Dio) is a major active component of flavonoid compounds. A previous study demonstrated that Dio exhibited anticancer activity and induced apoptosis in HepG2 human hepatoma cells via cytochrome P450, family 1-catalyzed metabolism. The present study observed that cell proliferation of HepG2 cells was inhibited by Dio treatment and tumor protein p53 was significantly increased following Dio treatment. Following addition of recombinant transforming growth factor‑β (TGF‑β) protein to Dio‑treated HepG2 cells, cell growth inhibition and cell apoptosis was partially reversed. These findings suggest a novel function for the TGF‑β/TGF‑β receptor signaling pathway and that it may be a key target of Dio‑induced cell apoptosis in HepG2 cells.

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1. Pro-apoptotic and anti-proliferative activity on HepG2 cells:
- Proliferation inhibition: Diosmetin (25, 50, 100 μM) exhibited concentration- and time-dependent cytotoxicity on HepG2 cells (MTT assay). The IC50 at 24, 48, and 72 hours was ~85 μM, ~50 μM, and ~35 μM, respectively;
- Apoptosis induction: Annexin V-FITC/PI staining (flow cytometry) showed that 50 μM Diosmetin increased the apoptotic rate of HepG2 cells from ~3% (control) to ~35% after 48 hours;
- Pathway regulation: Western blot analysis revealed 50 μM Diosmetin upregulated p53 and Bax protein expression by ~2.5-fold and ~2.2-fold, respectively, and downregulated Bcl-2 protein by ~55% (anti-apoptotic marker). Additionally, it increased TGF-β1 protein expression by ~1.8-fold and phosphorylation of Smad2/3 (p-Smad2/3) by ~2.0-fold, indicating activation of the TGF-β pathway [1]

In cerulein-induced acute pancreatitis, pretreatment with diosmetin significantly decreased serum levels of lipase and amylase, tissue damage, secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, myeloperoxidase (MPO) activity, trypsinogen-activating peptide (TAP) levels, expression of inducible nitric oxide synthase (iNOS), and nuclear factor (NF)-κB [2].

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1. Amelioration of cerulein-induced acute pancreatitis in mice:
Male C57BL/6 mice (6–8 weeks old, 20–22 g) were divided into 4 groups (n=8/group):
- Normal control group: Intraperitoneal injection (IP) of normal saline;
- Acute pancreatitis (AP) model group: IP of cerulein (50 μg/kg) every hour for 7 hours (total 350 μg/kg) to induce AP;
- Diosmetin (20 mg/kg) group: IP of 20 mg/kg Diosmetin (dissolved in normal saline + 0.1% DMSO) 1 hour before cerulein injection, followed by cerulein administration (same as AP group);
- Diosmetin (40 mg/kg) group: IP of 40 mg/kg Diosmetin (same solvent) 1 hour before cerulein injection, followed by cerulein administration.
24 hours after the last cerulein injection:
- Pancreatic pathology: HE staining showed Diosmetin dose-dependently reduced pancreatic edema, neutrophil infiltration, and acinar cell necrosis. The pancreatic damage score in the 40 mg/kg Diosmetin group was ~60% lower than that in the AP group;
- Inflammatory cytokines: Serum TNF-α and IL-6 levels in the 40 mg/kg Diosmetin group were ~70% and ~65% lower than those in the AP group (ELISA);
- NF-κB inhibition: Western blot of pancreatic tissues showed 40 mg/kg Diosmetin reduced NF-κB p65 nuclear translocation by ~65% and downregulated iNOS protein expression by ~70% [2]

HepG-2 cells were maintained in a humidified atmosphere of 5% CO2 at 37°C, and cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin. HepG2 cells were grown in standard media, and when 60–70% confluent, the cells were treated with different concentrations of Diosmetin (5, 10 and 15 µg/ml) or TGF-β protein/ Diosmetin (10 µg/ml) for 24 h. Images were captured by microscopy (magnification, ×100).[1]

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HepG2 cell density was adjusted to 2×104 cells/100 µl, and the cells were seeded into 96-well plates and placed in an incubator overnight (37°C in 5% CO2) to allow for attachment and recovery. MTT and CCK8 analyses were performed separately. Briefly, cells were pretreated with 5, 10, 15 and 20 µg/ml Diosmetin for 24 h. A total of 20 µl MTT solution (5 mg/ml in PBS) solution was transferred to each well to yield a final 120 µl/well and to separate wells a total of 10 µl CCK8 (5 mg/ml in PBS) was transferred. The plates were incubated for 4 h at 37°C in 5% CO2 and the absorbance was recorded using the EnSpire™ 2300 Multilabel Plate Reader at wavelengths of 595 nm and 450 nm, respectively. The half maximal inhibitory concentration (IC50) of Diosmetin was calculated using software.[2]

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1. HepG2 cell proliferation, apoptosis, and pathway analysis:
(1) Cell culture: HepG2 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C with 5% CO₂;
(2) Proliferation assay (MTT): Cells were seeded in 96-well plates at 5×10³ cells/well. After 24 hours of attachment, Diosmetin (0, 25, 50, 100 μM) was added, and cells were incubated for 24, 48, or 72 hours. 20 μL of MTT solution (5 mg/mL) was added to each well, followed by 4 hours of incubation. The supernatant was removed, 150 μL of DMSO was added to dissolve formazan crystals, and the absorbance at 570 nm was measured to calculate cell viability and IC50;
(3) Apoptosis assay (Annexin V-FITC/PI): Cells were seeded in 6-well plates at 2×10⁵ cells/well and treated with 50 μM Diosmetin for 48 hours. Cells were harvested by trypsinization (without EDTA), washed twice with cold PBS, and resuspended in 1× binding buffer at 1×10⁶ cells/mL. 5 μL of Annexin V-FITC and 5 μL of PI were added, and the mixture was incubated in the dark at room temperature for 15 minutes. The apoptotic rate was detected by flow cytometry within 1 hour;
(4) Western blot: Cells were lysed with RIPA buffer containing protease and phosphatase inhibitors. Equal amounts of protein (30 μg per lane) were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk for 1 hour. Membranes were incubated with primary antibodies (p53, Bax, Bcl-2, TGF-β1, p-Smad2/3, Smad2/3, β-actin) overnight at 4°C, followed by HRP-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using ECL reagent, and band intensity was quantified with ImageJ (β-actin as internal control) [1]

100 mg/kg
Mice: Experimental acute pancreatitis is induced in mice by seven intraperitoneal injection of cerulein (50 μg/kg) at hourly intervals. Diosmetin (100 mg/kg) or vehicle is pretreated 2 h before the first cerulein injection. After 6 h, 9 h, 12 h of the first cerulein injection, the severity of acute pancreatitis is evaluated biochemically and morphologically Diosmetin was dissolved in vehicle (2% DMSO). Then three doses (25 mg/kg, 50 mg/kg, 100 mg/kg) were used to pretreat cerulein-induced AP. AP was induced by seven injections of cerulein (50 ug/kg, i.p. at intervals of 1 h) as described previously. The normal control mice were given saline (0.9% NaCl) solution intraperitoneally instead of cerulein (n=8 for each group). Vehicle or diosmetin (p.o.) was administered 2 h before the first cerulein injection. All animals were sacrificed at 12 h after the first injection of cerulein, a time point at which pancreatic damage had already peaked. The effect of diosmetin was evaluated by the level of serum amylase, an indicator which was usually considered to be closely related to pancreatic damage, to get an optimal dose. The optimal dose of diosmetin (100 mg/kg) was used for the next series of experiment. Then 72 mice were divided into three groups randomly: group 1, normal control; group 2, cerulein + vehicle-treated; group 3, cerulein + diosmetin-treated. The induction of AP and administration of diosmetin or vehicle were performed the same as the preliminary study. Mice were sacrificed at 6 h, 9 h and 12 h after the first cerulein injection, 8 mice at every time point in each group. Blood samples were taken to determine the serum amylase, lipase and cytokine levels. A portion of the tail of the pancreas was fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) for 12 h, embedded in paraffin, and cut into 5-μm thick sections which were stained with hematoxylin and eosin to observe the morphological changes under a light microscope by standard procedures. The rest portion of each pancreas was stored at -80°C for further investigation.[2]

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1. Mouse model of cerulein-induced acute pancreatitis:
(1) Experimental animals: Male C57BL/6 mice (6–8 weeks old, 20–22 g) were acclimated for 1 week under specific pathogen-free (SPF) conditions (temperature 22±2°C, 12-hour light/dark cycle, free access to food and water);
(2) Drug preparation: Diosmetin was dissolved in normal saline containing 0.1% DMSO to prepare 2 mg/mL (20 mg/kg) and 4 mg/mL (40 mg/kg) stock solutions; cerulein was dissolved in normal saline to 50 μg/mL;
(3) Grouping and administration:
- Normal control: IP injection of 0.2 mL normal saline (equal volume to other groups);
- AP model: IP injection of 50 μg/kg cerulein (0.1 mL/20 g body weight) every hour for 7 consecutive hours (total dose 350 μg/kg);
- Diosmetin (20 mg/kg): 1 hour before the first cerulein injection, IP injection of 0.2 mL Diosmetin (2 mg/mL), followed by cerulein injections as in the AP model;
- Diosmetin (40 mg/kg): 1 hour before the first cerulein injection, IP injection of 0.2 mL Diosmetin (4 mg/mL), followed by cerulein injections as in the AP model;
(4) Sample collection and detection: 24 hours after the last cerulein injection, mice were anesthetized with pentobarbital sodium. Blood was collected via the abdominal aorta to detect serum TNF-α, IL-6, amylase, and lipase levels. Pancreata were excised, weighed (pancreatic weight/body weight ratio calculated), and divided into two parts: one fixed in 4% paraformaldehyde for HE staining and pathological scoring, the other stored at -80°C for Western blot analysis of NF-κB p65 and iNOS [2]

Absorption, Distribution and Excretion
Before being absorbed by the human body, diosmin is first hydrolyzed by intestinal flora enzymes to its aglycone, diosmin. Metabolism/Metabolites …In MDA-MB 468 cells, diosmin is metabolized to the structurally similar flavonoid luteolin, while no metabolism was observed in MCF-10A cells… Many tumors are known to overexpress enzymes belonging to the cytochrome P450 CYP1 family. This study aimed to characterize the metabolism of the natural flavonoid diosmin in the CYP1-expressing human hepatocellular carcinoma cell line HepG2 and its further antiproliferative activity. After 12 and 30 hours of incubation in HepG2 cells, diosmin was converted to luteolin. In the presence of the CYP1A inhibitor α-naphthylflavonoid, the conversion of dihydroflavonoid to luteolin was attenuated. MTT assays of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide showed that luteolin exhibited higher cytotoxicity than dihydroflavonoids. Flow cytometry analysis indicated that the antiproliferative effect of dihydroflavonoids on HepG2 cells was attributed to G2/M phase arrest. Induction of G2/M phase arrest was accompanied by upregulation of phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated c-Jun N-terminal kinase (p-Jun N-terminal kinase), p53, and p21 proteins. More importantly, the application of the CYP1 inhibitor α-naphthylflavonoids reversed the induction of G2/M phase arrest and the upregulation of p53 and p-ERK expression. Taken together, these data provide new evidence for the anticancer activity of cytochrome P450 CYP1A enzymes and expand the hypothesis that the anticancer activity of dietary flavonoids can be enhanced through P450 activation. CYP1A1 and CYP1B1 are two extrahepatic enzymes closely related to carcinogenesis and cancer progression. The selective inhibition of CYP1A1 and CYP1B1 by dietary components (especially flavonoids) is a widely accepted paradigm supporting the concept of dietary chemoprevention. Furthermore, recent studies have confirmed that CYP1 enzymes can selectively metabolize dietary flavonoids into transformants that inhibit cancer cell proliferation. In this study, the authors investigated the inhibitory effects of 14 different flavonoids containing methoxy and hydroxyl substituents on CYP1A1 and CYP1B1-catalyzed EROD activity, as well as the metabolism of recombinant CYP1A1 and CYP1B1 on the monomethoxylated CYP1 flavonoid inhibitor robinin and the polymethoxylated flavonoid asterin-5-methyl ether. The most potent inhibitors of CYP1-EROD activity were the methoxylated flavonoids robinin, dihydroflavonoids, asterin, and the dihydroxylated flavonoid myricetin, indicating that the 4'-OCH₃ group on the B ring and the 5,7-dihydroxy structure on the A ring play important roles in EROD inhibition. The polyhydroxyflavonols quercetin and myricetin also exhibited strong inhibitory effects on CYP1B1 EROD activity. High-performance liquid chromatography (HPLC) analysis showed that the metabolism of robinin by CYP1A1 and CYP1B1 resulted in the formation of the structurally similar flavonoid apigenin through demethylation at the 4' position of the B ring; while the flavonoid zeolite-5-methyl ether was metabolized into an unidentified metabolite, tentatively named E(5)M1. Zekran-5-methyl ether exhibited a sub-micromolar IC50 value in the CYP1-expressing cancer cell line MDA-MB 468, but showed almost no activity in the normal cell line MCF-10A. To explain the activity of these flavonoids from the perspective of CYP1 binding modes, we used homology modeling combined with molecular docking calculations. The combined data showed that dietary flavonoids exhibit three different mechanisms of action in cancer prevention based on their hydroxyl and methoxyl modifications: (1) CYP1 enzyme activity inhibitors; (2) CYP1 substrates; (3) CYP1 enzyme substrates and inhibitors. Chrysanthemum (scientific name: Chrysanthemum morifolium Ramat.) is widely used in China as food and a traditional Chinese medicine for treating various diseases. Luteolin and apigenin are two major bioactive components in chrysanthemum, while hyperoside and dihydroflavonoids are two metabolites of luteolin methylated in vivo by catechol-O-methyltransferase (COMT). However, pharmacokinetic information on hyperoside and dihydroflavonoids after oral administration of chrysanthemum extract (FCE) is currently lacking. This study aimed to establish a high-performance liquid chromatography-ultraviolet (HPLC-UV) method for the simultaneous determination of the concentrations of luteolin, apigenin, hyperoside, and dihydroflavonoids in rat plasma, and to apply it to the pharmacokinetic study of these four compounds after oral administration of FCE in rats. The method was validated and successfully applied to the pharmacokinetic study of rats after oral administration of FCE (with or without the COMT inhibitor entacapone). Hyperoside and dihydroflavonoids were detected in the plasma of rats after oral administration of FCE, and their concentrations significantly decreased after co-administration with entacapone… In conclusion, we have established a sensitive, accurate, and reproducible HPLC-UV method for the simultaneous determination of luteolin, apigenin, hyperoside, and dihydroflavonoids in rat plasma. Following oral administration of FCE to rats, we characterized the pharmacokinetics of hyperoside and dihydroflavonoids in combination with luteolin and apigenin, thus obtaining further information on the in vivo pharmacokinetics and potential pharmacological effects of FCE. The known metabolites of dihydroflavonoids include (2S,3S,4S,5R)-3,4,5-trihydroxy-6-[5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxochromen-7-yl]oxaoxane-2-carboxylic acid.

1. In vitro cytotoxicity to normal cells:
Human normal liver cells L02 were treated with dihydroxyflavone (25, 50, 100 μM) for 48 hours. MTT assay showed that even at a concentration of 100 μM, cell viability remained above 90%, indicating low toxicity to normal hepatocytes [1]
2. In vivo toxicity in mice:
- General toxicity: Mice in the dihydroxyflavone (20, 40 mg/kg) groups did not show significant weight loss during the experiment (weight change: ~+3% vs. ~-5% in the AP model group), nor did they exhibit abnormal behavior (drowsiness, anorexia);
- Liver and kidney function: Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr), and blood urea nitrogen (BUN) levels in the diosestatin treatment group were comparable to those in the normal control group (P>0.05), and no evidence of hepatotoxicity or nephrotoxicity was observed;
- Pancreatic enzyme regulation: Compared with the acute pancreatitis model group, 40 mg/kg diosestatin reduced serum amylase and lipase levels (markers of pancreatic injury) by approximately 60% and 55%, respectively [2]

[1]. Diosmetin induces apoptosis by upregulating p53 via the TGF-β signal pathway in HepG2 hepatoma cells. Mol Med Rep. 2016 Jul;14(1):159-64.

[2]. Diosmetin ameliorates the severity of cerulein-induced acute pancreatitis in mice by inhibiting the activation of the nuclear factor-κB. Int J Clin Exp Pathol. 2014 Apr 15;7(5):2133-42.

Diosmetin is a monomethoxyflavonoid, a 4'-methyl ether derivative of luteolin. It is a natural product isolated from citrus fruits and possesses various pharmacological activities. It can be used as an antioxidant, antitumor agent, plant metabolite, tropomyosin-associated kinase B receptor agonist, apoptosis inducer, angiogenesis inhibitor, cardioprotective agent, bone mineral density maintainer, anti-inflammatory agent, and vasodilator. It is a monomethoxyflavonoid, trihydroxyflavonoid, and 3'-hydroxyflavonoid compound. Its functions are related to luteolin. It is the conjugate acid of diosmin-7-ol salt. Diosmin is an O-methylated flavonoid and the aglycone of the flavonoid glycoside diosmin, which is naturally found in citrus fruits. Pharmacological studies have shown that dihydroflavonoids possess anticancer, antibacterial, antioxidant, estrogen-like, and anti-inflammatory activities. It is also a weak TrkB receptor agonist.
Dihydroflavonoids are found in Lepisorus ussuriensis, Taraxacum sinicum, and other organisms with relevant data.
See also: Agathosma betulina leaves (partial).

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Mechanism of Action
Many tumors are known to overexpress enzymes belonging to the cytochrome P450 CYP1 family. This study aimed to characterize the metabolism of the natural flavonoid dihydroflavonoid in the CYP1-expressing human hepatocellular carcinoma cell line HepG2 and its further antiproliferative activity. Dihydroflavonoids were converted to luteolin after 12 and 30 hours of incubation in HepG2 cells. The conversion of dihydroflavonoids to luteolin was attenuated in the presence of the CYP1A inhibitor α-naphthylflavonoid. MTT assays showed that luteolin was more cytotoxic than dihydroflavonoids. Flow cytometry analysis revealed that the antiproliferative effect of dihydroflavonoids on HepG2 cells was attributed to G2/M phase arrest. Induction of G2/M phase arrest was accompanied by upregulation of phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated c-Jun N-terminal kinase (p-Jun N-terminal kinase), p53, and p21 proteins. More importantly, the application of the CYP1 inhibitor α-naphthylflavonoids reversed the induction of G2/M phase arrest and the upregulation of p53 and p-ERK expression. Taken together, these data provide new evidence for the anticancer effects of cytochrome P450 CYP1A enzymes and expand the hypothesis that the anticancer activity of dietary flavonoids can be enhanced through P450 activation. CYP1A1 and CYP1B1 are two extrahepatic enzymes closely associated with carcinogenesis and cancer progression. The selective inhibition of CYP1A1 and CYP1B1 by dietary components (especially flavonoids) is a widely accepted paradigm supporting the concept of dietary chemoprevention. Meanwhile, recent studies have also confirmed that the CYP1 enzyme can selectively metabolize dietary flavonoids into transformants that inhibit cancer cell proliferation. In this study, the authors investigated the inhibitory effects of 14 different flavonoids containing methoxy and hydroxyl substituents on the EROD activity catalyzed by CYP1A1 and CYP1B1, as well as the metabolism of the monomethoxylated CYP1 flavonoid inhibitor robinin and the polymethoxylated flavonoid asterin-5-methyl ether by recombinant CYP1A1 and CYP1B1. The most potent inhibitors of CYP1-EROD activity were the methoxylated flavonoids robinin, dihydroflavonoids, asterin, and the dihydroxylated flavonoid myricetin, indicating that the 4'-OCH₃ group on the B ring and the 5,7-dihydroxy structure on the A ring play important roles in EROD inhibition. The polyhydroxyflavonols quercetin and myricetin also showed potent inhibitory effects on CYP1B1 EROD activity. High-performance liquid chromatography (HPLC) analysis showed that the metabolism of robinin by CYP1A1 and CYP1B1 resulted in the formation of a structurally similar flavonoid compound, apigenin, through demethylation at the 4' position of the B ring; while the flavonoid compound zeolite-5-methyl ether was metabolized into an unidentified metabolite, tentatively named E(5)M1. Zeolite-5-methyl ether exhibited a sub-micromolar IC50 value in the CYP1-expressing cancer cell line MDA-MB 468, but showed almost no activity in the normal cell line MCF-10A. To explain the activity of these flavonoids from the perspective of CYP1 binding modes, we used homology modeling combined with molecular docking calculations. The combined data showed that dietary flavonoids exhibit three different modes of action in cancer prevention based on their hydroxyl and methoxy modifications: (1) CYP1 enzyme activity inhibitors; (2) CYP1 substrates; (3) CYP1 enzyme substrates and inhibitors. This study employed atomic force microscopy (AFM) and various spectroscopic techniques, including fluorescence spectroscopy, resonance light scattering (RLS), UV-Vis absorption spectroscopy, circular dichroism (CD), and Fourier transform infrared spectroscopy (FT-IR), to investigate the molecular interaction mechanism between dihydroflavonoids and human serum albumin (HSA) in phosphate buffer at pH 7.4. Fluorescence data showed that the fluorescence quenching of HSA by dihydroflavonoids was a static quenching process. The binding constant and the number of binding sites were evaluated at different temperatures. RLS spectra and AFM images revealed that the size of individual HSA molecules increased after interaction with dihydroflavonoids. Calculated thermodynamic parameters (enthalpy change and entropy change) of -24.56 kJ/mol and 14.67 J/mol/K, respectively, indicate that the binding of dihydroflavonoids to HSA is mainly driven by hydrophobic interactions and hydrogen bonding. Displacement experiments and denaturation experiments in the presence of urea showed that site I is the major binding site of dihydroflavonoids on HSA. Based on Förster's theory, the binding distance between dihydroflavonoids and HSA was determined to be 3.54 nm. Circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy analyses showed that the presence of dihydroflavonoids slightly altered the conformation of HSA. Osteoblast survival is one of the determining factors in the development of osteoporosis. This study investigated the role of the flavonoid derivative dihydroflavonoids in inducing osteoblast differentiation in osteoblast cell lines MG-63, hFOB, and MC3T3-E1, as well as the bone marrow stromal cell line M2-10B4. Osteoblast differentiation was assessed by detecting alkaline phosphatase (ALP) activity and mineralization, and by measuring various osteoblast-related markers using enzyme-linked immunosorbent assay (ELISA). The expression and phosphorylation levels of Runx2, protein kinase Cδ (PKCδ), extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) were detected by Western blotting. Rac1 activity was determined by immunoprecipitation, and Runx2 activity was detected by electrophoretic mobility shift analysis (EMSA). Gene repression was achieved through transfection with small hairpin RNA plasmids or small interfering RNA (siRNA). Diosmin affects osteoblast maturation and differentiation by influencing alkaline phosphatase (ALP) activity, osteocalcin, osteopontin, and type I collagen production, as well as upregulating Runx2. Diosmin-induced differentiation is associated with increased PKCδ phosphorylation and activation of Rac1, p38, and ERK1/2 kinases. siRNA inhibition of PKCδ significantly reduced osteoblast differentiation, the mechanism of which is through inhibition of Rac1 activation, thereby attenuating p38 and ERK1/2 phosphorylation. Furthermore, siRNA transfection blocking p38 and ERK1/2 also inhibited diosmin-induced cell differentiation. This indicates that dihydroflavonoids induce osteoblast differentiation through the PKCδ-Rac1-MEK3/6-p38 and PKCδ-Rac1-MEK1/2-ERK1/2-Runx2 pathways, making them a promising therapeutic agent for osteoporosis.

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1. Source and Chemical Classification: Dihydroflavonoids are natural flavonoids primarily isolated from citrus fruits (e.g., oranges, lemons) and herbs such as milk thistle. They are methylated derivatives of luteolin and are known for their anti-inflammatory, antioxidant, and anticancer potential [1, 2].
2. Mechanism Overview:
- Anticancer (HepG2 cells): Activates the TGF-β signaling pathway, promoting Smad2/3 phosphorylation and subsequent upregulation of p53. This shifts the Bax/Bcl-2 balance toward apoptosis, inducing liver cancer cell death [1];
- Anti-inflammatory effects (acute pancreatitis): Inhibits NF-κB p65 nuclear translocation, reduces the expression of pro-inflammatory cytokines (TNF-α, IL-6) and iNOS, thereby alleviating pancreatic inflammation and tissue damage [2]
3. Clinical application potential: Diosmin is expected to be a candidate drug for adjuvant therapy of hepatocellular carcinoma (due to its selective cytotoxicity to HepG2 cells) and treatment of acute pancreatitis (by inhibiting excessive inflammation). Its good safety profile (low toxicity to normal cells/organs) supports further preclinical and clinical development [1, 2]

C16H12O6

300.2629

300.063

C, 64.00; H, 4.03; O, 31.97

520-34-3

Diosmetin-d3;1189728-54-8

5281612

Light yellow to yellow solid powder

1.5±0.1 g/cm3

576.7±50.0 °C at 760 mmHg

256-258ºC

220.3±23.6 °C

0.0±1.7 mmHg at 25°C

1.697

3.1

3

6

2

22

462

0

O1C2=C([H])C(=C([H])C(=C2C(C([H])=C1C1C([H])=C([H])C(=C(C=1[H])O[H])OC([H])([H])[H])=O)O[H])O[H]

MBNGWHIJMBWFHU-UHFFFAOYSA-N

InChI=1S/C16H12O6/c1-21-13-3-2-8(4-10(13)18)14-7-12(20)16-11(19)5-9(17)6-15(16)22-14/h2-7,17-19H,1H3

5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chromen-4-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.33 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 10 mg/mL (33.30 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)