Apoptosis; Bcl-2; Bcl-xL

Dihydrokaempferol (0.3–300 μM; 48 hours) does not appear to affect normal synoviocytes, however it is necessary to lower RA-FLS activity[1]. In a 48-hour period, Dihydrokaempferol (3-30 μM) increases the number of dry cells (containing early and late plaque cells; approximately 3.8% and 9.6%, respectively) [1]. Over the course of 48 hours, dihydrokaempferol (3–30 μM) enhances the expression of Bax and Bad, suppresses the expression of Bcl-2 and Bcl-xL.

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In addition, apoptosis effects of abundant Dihydrokaempferol were evaluated in vitro. Dihydrokaempferol exhibited inhibitory effects on the proliferation of synoviocytes. Furthermore, dihydrokaempferol promoted Bax and Bad expression, as well as the cleavage of caspase-9, caspase-3, and PARP. Meanwhile, it inhibited Bcl-2 and Bcl-xL expression. These findings indicate that dihydrokaempferol isolated from the ethyl acetate extract of B. championii effectively promotes apoptosis, which is an important process through suppression of apoptotic activity. The results are encouraging for further studies on the use of B. championii in the treatment of RA.

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Dihydrokaempferol Decreases the Proliferation of RA-FLSs [1]
As illustrated in Figure 2, Dihydrokaempferol (0.3, 3, 30, 300 μM) had no significant effect of cell survival on normal synoviocytes (Figure 2(a)). But dihydrokaempferol (0.3, 3, 30, 300 μM) concentration dependently decreased the viability of RA-FLSs (Figure 2(b)). Treatment of these cells with more than 3 µM concentration of dihydrokaempferol for 48 h resulted in significant decrease of cell viability (Figure 2(b)). In light of these findings, we used 3, 30 µM concentration of dihydrokaempferol for our subsequent experiments.

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Dihydrokaempferol Induces Apoptosis in RA-FLSs [1]
To confirm whether Dihydrokaempferol induced apoptosis in RA-FLSs, the annexin V/PI double staining assay was performed. It examined the reversion of phosphatidylserine (a marker for apoptosis) by flow cytometric analysis. As demonstrated in Figure 3, following treatment with dihydrokaempferol (3, 30 µM), the percentage of apoptotic cells (including early and late apoptotic cells) was found gradually increased (~3.8% and ~9.6%, respectively) as compared to control treatment (~2.3%). It was suggested that dihydrokaempferol significantly induced apoptosis in RA-FLSs.

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Dihydrokaempferol Regulated the Protein Expression of Apoptosis in RA-FLSs [1]
In further part of the study, cells were incubated with different concentrations of Dihydrokaempferol to evaluate its proapoptotic activity toward synovial cells. The result (Figure 4) showed that dihydrokaempferol significantly promoted Bax and Bad expression and inhibited Bcl-2 and Bcl-xL expression. Moreover, as shown in Figure 5, the cleaved fragments of caspase-3 and caspase-9 were significantly increased by dihydrokaempferol, and the protein level of cleaved PARP was markedly increased as well. Collectively these findings indicated that dihydrokaempferol instigated apoptosis.

MDA as a lipid peroxidation marker, was increased over 3 folds in TAC-treated mice hearts. The increase was effectively suppressed by ARO (Aromadendrin)/Dihydrokaempferol in vivo (Fig. 3A). Another lipid peroxidation marker 4-HNE was increased after TAC procedures, and ARO down-regulated this trend (Fig. 3B and C). Likewise, the GSH/GSSG ratio is an important anti-antioxidant marker. Herein, this study noticed a reduced GSH/GSSG ratio in TAC-treated mice and ARO restored the decrease (Fig. 3D) in vivo. The cell-permeant DCF-DA assay confirmed that ARO had no effects on the ROS under basal condition, while substantially decrease of ROS formation after PE treatment in a concentration-related pattern (Fig. 3E). [2]

cell proliferation analysis [1]
Cell Types: normal synoviocytes; RA-FLS cells
Tested Concentrations: 0.3 μM, 3 μM, 30 μM, 300 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: diminished proliferation of RA-FLS.

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Apoptosis analysis [1]
Cell Types: RA-FLS Cell
Tested Concentrations: 3 μM, 30 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Induced RA-FLS cell apoptosis.

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Western Blot Analysis [1]
Cell Types: RA-FLS Cell
Tested Concentrations: 3 μM, 30 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Promote Bax and Bad expression, increase caspase-3, caspase-9 and cleaved PARP fragments and inhibit Bcl -2 and Bcl-xL expression.

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Cell Viability Assay [1]
RA-FLSs were cultured in 96-well plates and the cell viability was assessed by MTS assay. RA-FLSs cultured in 96-well plates were treated with Dihydrokaempferol at various concentrations (0.3, 3, 30, 300 μM) for 48 h, followed by incubation with MTS for an additional 4 h at 37°C. Then the absorbance at 570 nm was taken by a microplate reader.

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Flow Cytometric Analysis [1]
RA-FLSs cultured in 6-well plates were treated with Dihydrokaempferol at various concentrations for 48 h. Then, cells were harvested and quantitated according to the manufacture's protocol. Briefly, cells were resuspended in binding buffer and were incubated in 5 µL of annexin V-FITC and 5 µL of PI at room temperature for 15 min in the dark. Finally, 400 µL of binding buffer was added and then samples were analyzed by flow cytometer with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. Apoptotic cells were expressed as a percentage of the total number of cells and three times of flow cytometric analysis have been done.

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Western Blot Analysis [1]
According to the results of cell viability, western blot analysis was used to evaluate the proteins level affected by Dihydrokaempferol. Its method was similar to those described previously. After being treated with Dihydrokaempferol, cells were collected and lysed by lysis buffer, then they were centrifuged at 12,000 g for 15 min by Heraeus Sepatech. The supernatant was collected and the protein concentration was determined by the BCA method. Then protein mixed with loading buffer and incubated in 100°C for 6 min. Ultimately, samples were analyzed for western blot analysis with primary antibodies to cleaved caspase-3 (1:500), cleaved caspase-9 (1:500), p-Bad (1:500), Bcl-xL (1:1,000), Bax (1:1,000), Bcl-2 (1:1,000), cleaved PARP (1:1,000), and β-actin (1:1,000) overnight at 4°C.

[1]. Apoptosis Effects of Dihydrokaempferol Isolated from Bauhinia championii on Synoviocytes. Evid Based Complement Alternat Med. 2018 Dec 2;2018:9806160.

[2]. Inhibition of cardiac hypertrophy by aromadendrin through down-regulating NFAT and MAPKs pathways. Biochem Biophys Res Commun. 2018 Dec 2;506(4):805-811.

(+)-dihydrokaempferol is a tetrahydroxyflavanone having hydroxy groupa at the 3-, 4'-, 5- and 7-positions. It has a role as a metabolite. It is a tetrahydroxyflavanone, a member of dihydroflavonols, a secondary alpha-hydroxy ketone and a member of 4'-hydroxyflavanones. It is functionally related to a kaempferol. It is a conjugate acid of a (+)-dihydrokaempferol 7-oxoanion.
Aromadendrin has been reported in Camellia sinensis, Maclura pomifera, and other organisms with data available.
See also: Acai fruit pulp (part of).

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Bauhinia championii (Benth.) Benth. is a traditional medicinal plant used in China to treat rheumatoid arthritis (RA), especially in She ethnic minority group. This study focused on the active constituents from the rattan of B. championii (Benth.) Benth., which possess potential apoptosis effects. A conventional phytochemical separation method for the isolation of compounds from the ethyl acetate extract of B. championii was developed. The procedure involved extraction, liquid–liquid partitioning with ethyl acetate, and subsequent compound purification, respectively. Additionally, cell viability of dihydrokaempferol found abundantly in it was evaluated in vitro by MTS, and the antiapoptosis effect was evaluated by annexin V/PI staining (Flow Cytometry Analysis) and western blot. The results showed that nine flavonoids, and five other compounds, were isolated from the ethyl acetate extract of B. championii and were identified as β-sitosterol (1), 5,6,7,3',4',5'-hexamethoxyflavone (2), 3',4',5,7-tetrahydroxyflavone (3), 5,7,3',4',5'-pentamethoxyflavone (4), 4'-hydroxy-5,7,3',5'-pentamethoxyflavone (5), apigenin (6), liquiritigenin (7), 5, 7-dihydroxylcoumarin (8), 3',4',5,7, -pentamethoxyflavone (9), n-octadecanoate (10), lupine ketone (11), dibutylphthalate (12), dihydrokaempferol (13), and 5,7,3′,5′-tetrahydroxy-6-methylflavanone (14). Among these compounds, 5-14 were isolated for the first time from B. championii. In addition, apoptosis effects of abundant dihydrokaempferol were evaluated in vitro. Dihydrokaempferol exhibited inhibitory effects on the proliferation of synoviocytes. Furthermore, dihydrokaempferol promoted Bax and Bad expression, as well as the cleavage of caspase-9, caspase-3, and PARP. Meanwhile, it inhibited Bcl-2 and Bcl-xL expression. These findings indicate that dihydrokaempferol isolated from the ethyl acetate extract of B. championii effectively promotes apoptosis, which is an important process through suppression of apoptotic activity. The results are encouraging for further studies on the use of B. championii in the treatment of RA.[1]
Considering the obtained results, dihydrokaempferol and thirteen other compounds were isolated from the ethyl acetate extract of B. championii (Benth.) Benth. Given its recorded antiproliferative effects on synovial cells, dihydrokaempferols seem to be good candidates for new antiarthritic drugs and are recommended for further biomedical studies.[1]

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Cardiac hypertrophy is a maladaptive response to pressure overload and it's an important risk factor for heart failure and other adverse cardiovascular events. Aromadendrin (ARO) has remarkable anti-lipid peroxidation efficacy and is a potential therapeutic medicine for the management of diabetes and cardiovascular diseases. In this study, we established the cardiac hypertrophy cell model in rat neonatal ventricular cardiomyocytes (RNVMs) with phenylephrine. The cell model was characterized by the increased protein synthesis and cardiomyocyte size, which can be normalized by ARO treatment in both concentration- and time-dependent manner. In transverse aortic constriction (TAC) induced cardiac hypertrophy model, ARO administration improved the impairment of cardiac function and alleviated the cardiac hypertrophy indicators, like ventricular mass/body weight, myocyte cross-sectional area, and the expression of ANP, BNP and Myh7. ARO treatment also suppressed the cardiac fibrosis and the correlated fibrogenic genes. Our further investigation revealed ARO could down-regulate pressure overload-induced Malondialdehyde (MDA) and 4-HNE expression, restore the decrease of GSH/GSSG ratio, meanwhile prevent nuclear translocation of NFAT and the activation of MAPKs pathways. Collectively, ARO has a protective effect against experimental cardiac hypertrophy in mice, suggesting its potential as a novel therapeutic drug for pathological cardiac hypertrophy.[2]

C15H12O6

288.255

288.063

C, 62.50; H, 4.20; O, 33.30

480-20-6

480-20-6

122850

White to off-white solid powder

1.6±0.1 g/cm3

639.0±55.0 °C at 760 mmHg

247 - 249 °C

247.3±25.0 °C

0.0±2.0 mmHg at 25°C

1.729

2.42

4

6

1

21

392

2

O1C2=C([H])C(=C([H])C(=C2C([C@@]([H])([C@@]1([H])C1C([H])=C([H])C(=C([H])C=1[H])O[H])O[H])=O)O[H])O[H]

PADQINQHPQKXNL-LSDHHAIUSA-N

InChI=1S/C15H12O6/c16-8-3-1-7(2-4-8)15-14(20)13(19)12-10(18)5-9(17)6-11(12)21-15/h1-6,14-18,20H/t14-,15+/m0/s1

(2R,3R)-3,5,7-trihydroxy-2-(4-hydroxyphenyl)-2,3-dihydrochromen-4-one

Aromadendrin; trans-Dihydrokaempferol; dihydrokaempferol; 480-20-6; (+)-Dihydrokaempferol; katuranin; Aromadedrin; (+)-aromadendrin; Aromadendrol; trans Dihydrokaempferol; Dihydrokaempferol; (+) Aromadendrin; (+)-Aromadendrin; (+) Dihydrokaempferol; (+)-Dihydrokaempferol

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO: ~250 mg/mL (~867.3 mM)
H2O: < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.22 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 20.8 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: ≥ 2.08 mg/mL (7.22 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.22 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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