ROS/reactive oxygen species

After 24 and 48 hours of incubation, apigenin-7-glucoside demonstrated strong antiproliferative action on B16F10 melanoma cells. The number of cells in the subG0/G1, S, and G2/M phases increased as a result of apigenin-7-glucoside, whereas the fraction of cells in the G0/G1 phase significantly decreased. In B16F10 melanoma cells, apigenin-7-glucoside increases tyrosinase activity, melanogenesis, and production [1]. Specifically, apigenin 7-glucoside inhibits myeloid differentiation while inducing CD34+ cells to differentiate toward the erythroid lineage. Strong antioxidant action against reactive oxygen species (ROS) is exhibited by apigenin 7-glucoside in vitro in a concentration-dependent manner [2].

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Apigenin-7-glucoside, genkwanin and naringenin exhibited significant anti-proliferative activity against B16F10 melanoma cells after 24 and 48 h of incubation. Furthermore, apigenin-7-glucoside, genkwanin and naringenin provoked an increase of subG0/G1, S and G2/M phase cell proportion with a significant decrease of cell proportion in G0/G1 phases. The results evaluated using Hoechst 33,258, confirm that the percentage of B16F10 cells observed in the sub G0/G1 phase were undergoing apoptosis. Moreover, apigenin-7-glucoside and naringenin revealed an ability to enhance melanogenesis synthesis and tyrosinase activity of B16F10 melanoma cells. Whereas genkwanin induces a decrease of melanin synthesis by inhibiting tyrosinase activity. Significance: Our results promote the introduction of genkwanin in cosmetic preparations, as skin whitening agent, whereas apigenin-7-glucoside and naringenin should be introduced into cosmetic products as natural tanning agents. [1]

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The generation of blood cellular components from hematopoietic stem cells is important for the therapy of a broad spectrum of hematological disorders. In recent years, several lines of evidence suggested that certain nutrients, vitamins and flavonoids may have important roles in controlling the stem cell fate decision by maintaining their self-renewal or stimulating the lineage-specific differentiation. In this study, main olive leaf phytochemicals oleuropein (Olp), apigenin-7-glucoside (Api7G) and luteolin 7-glucoside (Lut7G) were investigated for their potential effects on hematopoietic stem cell differentiation using both phenotypic and molecular analysis. Oleuropein and the combination of the three compounds enhanced the differentiation of CD34+ cells into myelomonocytic cells and lymphocytes progenitors and inhibited the commitment to megakaryocytic and erythroid lineages. Treatment with Lut7G stimulated both the erythroid and the myeloid differentiation, while treatment with Api7G specifically induced the differentiation of CD34+ cells towards the erythroid lineage and inhibited the myeloid differentiation. Erythroid differentiation induced by Api7G and Lut7G treatments was confirmed by the increase in hemoglobin genes expressions (α-hemoglobin, β-hemoglobin and γ-hemoglobin) and erythroid transcription factor GATA1 expression. As revealed by microarray analysis, the mechanisms underlying the erythroid differentiation-inducing effect of Api7G on hematopoietic stem cells involves the activation of JAK/STAT signaling pathway. These findings prove the differentiation-inducing effects of olive leaf compounds on hematopoietic stem cells and highlight their potential use in the ex vivo generation of blood cells. [2]

Tyrosinase activity assay [1]
Tyrosinase enzyme activity was estimated by measuring rate of l-DOPA oxidation, as described previously with slight modification. Briefly, cells (106) were treated with different levels of apigenin-7-glucoside, genkwanin and naringenin for 48 h, the cells were then solubilized in phosphate buffer (0.1 M; pH 6.8) containing 0.1% Triton × 100. Lysate was clarified by centrifugation at 17,500 g for 10 min at 4 °C; 400 μl of supernatant was mixed with 400 μl of l-DOPA (0.15%), and absorbance was followed spectrophotometrically at 475 nm, every minute for 10 min, since the substrate added to the reaction mixture.

In this study, researchers have investigated the effects of apigenin-7-glucoside, genkwanin and naringenin, on mouse melanoma B16F10 cell proliferation. Influence of these natural products on percentage cell distribution in cycle phases and melanogenesis was also studied.
Cell viability was determined at various periods using the MTT assay, whereas effects of tested compounds on progression through the cell cycle were analyzed by flow cytometry. In addition, amounts of melanin and tyrosinase were measured spectrophotometrically at 475 nm. Besides, the mechanism involved on the death route induced by the tested molecules was evaluated using the bis-benzimide trihydrochloride coloration method (Hoechst 33258)[1].

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Cell cycle analysis using flow cytometry [1]
Mouse melanoma cells (B16F10 5 × 105 cells) were seeded into a 50 cm2 culture dish and incubated for 24 h. Cells were treated with different concentrations of compounds for 48 h, trypsinized and washed twice in PBS (pH = 7.4). Cells were harvested and incubated for 15 min at room temperature and washed twice in cold PBS (pH = 7.4). After treatment with Ribonuclease A (10 mg ml− 1) for 30 min at room temperature and staining with 50 ml propidium iodide (1 mg ml− 1) for 10 min, cell cycle analysis was conducted using FACS system (Beckman Coulter, Switzerland). Percentages of cells in each phase of the cell cycle were calculated.

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Quantitative fluorescence microscopy [1]
Cells were seeded into 96-well microtiter plates and 24 h later, serial dilution of the test samples were added to dedicated wells. The plates were then incubated 48 h before the cells in each well were harvested, washed with PBS, re-suspended in 1 ml PBS containing 10 μg bis-benzimide trihydrochloride (Hoechst 33,258), and incubated at room temperature for 15 min. Aliquots of the cells (10 μl) were then placed on glass slides, and triplicate samples of 100 cells each were counted and scored for incidence of apoptotic chromatin condensation using a fluorescent microscope (Zeiss, Oberkochen, Germany). Stained nuclei with condensed chromatin (super-condensed chromatin at nuclear periphery) or nuclei that were fragmented into multiple smaller dense bodies were considered as apoptotic. Nuclei with uncondensed and dispersed chromatin were considered not apoptotic.

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Measurement of cellular melanin content [1]
Melanin release by cells was measured as described in Skandrani et al. Briefly, B16F10 cells (5 × 105) were seeded into a 50 cm2 culture dish with 10 ml of culture medium, and incubated for 24 h. Then, cells were treated with different concentration of aapigenin-7-glucoside, genkwanin and naringenin for 48 h. After treatment, melanogenesis activity (closely related to the amount of produced melanin), was estimated from amount of melanin secreted into cultured medium (extracellular melanin). Adherent cells were detached by incubation in trypsin (2.5%). Cells were then placed in tubes and solubilized in 1 ml of Triton × 100 (0.1%). Spectrophotometric absorbance of extracellular melanin content was measured at 475 nm. Absorbance was compared against a standard curve of known concentration of synthetic melanin and amounts estimated.

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The individual effect of each compound was assessed by treatment with Olp, Api7G/apigenin-7-glucoside or Lut7G at a final concentration of 50 µM based on a preliminary study of the morphology and the viability of CD34+ cells cultured in the presence of different concentrations of each compound (Samet et al., 2014b). DMSO (vehicle) at 0.05% was added to control cells. In order to evaluate the effect of the combination of these three compounds, cells were also treated with combination (comb) of Olp, Api7G and Lut7G at 55 µM, 5 µM, and 5 µM, respectively. The concentrations of the compounds in the mixture were determined based on the HPLC analysis of the ethanol extract of olive leaf and the compounds’ effective concentration for the induction of differentiation of human chronic myelogenous leukemia K562 cells in our previous study (Samet et al., 2014a).[2]

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Cell viability and cell number [2]
The viability and number of CD34+ cells were determined using flow cytometry on the 3rd, 6th and 9th days of culture with each compound (Olp, Api7G/apigenin-7-glucoside or Lut7G) or their combination (Comb). DMSO-treated CD34+ cells served as control cells. After incubation for the indicated time, treated cells were harvested, suspended in Guava ViaCount reagent and allowed to be stained for at least 5 min in darkness. The Guava ViaCount reagent contains two DNA-binding dyes. The nuclear dye stains only nucleated cells, while the viability dye brightly stains dying cells. The differential permeability of these two dyes enables the Guava ViaCount assay to distinguish between viable and non-viable cells. The cell number and viability were automatically measured using the Guava ViaCount application in Guava PCA flow cytometry. Morphological changes were detected by observation under a phase contrast microscope.

[1]. Effect of apigenin-7-glucoside, genkwanin and naringenin on tyrosinase activity and melanin synthesis in B16F10 melanoma cells. Life Sci. 2016 Jan 1;144:80-5.

[2]. Olive leaf components apigenin 7-glucoside and luteolin 7-glucoside direct human hematopoietic stem cell differentiation towards erythroid lineage. Differentiation. 2015 Jun;89(5):146-55.

Apigenin 7-O-beta-D-glucoside is a glycosyloxyflavone that is apigenin substituted by a beta-D-glucopyranosyl moiety at position 7 via a glycosidic linkage. It has a role as a non-steroidal anti-inflammatory drug, a metabolite and an antibacterial agent. It is a beta-D-glucoside, a dihydroxyflavone, a glycosyloxyflavone and a monosaccharide derivative. It is functionally related to an apigenin. It is a conjugate acid of an apigenin 7-O-beta-D-glucoside(1-). It is an enantiomer of an apigenin 7-O-beta-L-glucoside.
Cosmosiin has been reported in Acanthus ebracteatus, Coreopsis lanceolata, and other organisms with data available.
See also: Chamomile (part of).

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In summary, this study suggests that apigenin-7-glucoside and naringenin have a potential to be used as a natural product for tanning in cosmetic applications. We also report that genkwanin might be a useful therapeutic agent in the treatment of hyperpigmentation and provides effective component in skin-whitening cosmetics, since there is a concerted effort to search for naturally occurring tyrosinase inhibitors from plant because plants constitute a rich source of bioactive chemicals and many of them are largely free from harmful adverse effect. Moreover, apigenin-7-glucoside, genkwanin and naringenin inhibited population growth of mouse melanoma B16F10 cells in a dose-dependent manner. Further investigations are required to understand molecular mechanisms by which apigenin-7-glucoside, genkwanin and naringenin affect the cell cycle, before drawing final conclusions concerning mechanisms of the probable anti-cancer effects of the tested compounds. Although further studies are necessary to understand molecular mechanisms underlying the differentiating effect induced by apigenin-7-glucoside, genkwanin and naringenin on B16F10 cells, our results provide a new perspective for development of novel strategies for prevention and treatment of melanoma. [1]

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This study provides the first report on the differentiation-inducing effects of olive leaf phytochemicals on human hematopoietic stem cells. The described effects highlight the potential use of olive leaf components in optimizing the ex vivo generation of the selected hematopoietic sub-populations. Addition of apigenin-7-glucoside/Api7G in particular as a supplement to the culture medium, specifically induces the differentiation of CD34+ cells towards the erythroid lineage despite the absence of EPO. Our findings suggest the involvement of the JAK/STAT pathway. A more in depth analysis of the different components of JAK/STAT pathway will be carried out in the future at both gene and protein levels. Furthermore, the potential synergistic effects of flavonoid Api7G and the lineages specific-cytokine EPO on erythroid differentiation will be investigated. It will be also interesting to evaluate the effect of this flavonoid on the engraftment potential of the ex vivo generated hematopoietic cells in NOD/SCID mice. [2]

C21H20O10

432.3775

432.105

C, 58.34; H, 4.66; O, 37.00

578-74-5

Apigenin;520-36-5

5280704

Light yellow to yellow solid powder

1.6±0.1 g/cm3

788.9±60.0 °C at 760 mmHg

230-237ºC

280.7±26.4 °C

0.0±2.9 mmHg at 25°C

1.717

-0.39

6

10

4

31

675

5

C1=CC(=CC=C1C2=CC(=O)C3=C(C=C(C=C3O2)O[C@H]4[C@@H]([C@H]([C@@H]([C@H](O4)CO)O)O)O)O)O

KMOUJOKENFFTPU-OBJCFNGXSA-N

1S/C21H20O10/c22-8-16-18(26)19(27)20(28)21(31-16)29-11-5-12(24)17-13(25)7-14(30-15(17)6-11)9-1-3-10(23)4-2-9/h1-7,16,18-24,26-28H,8H2/t16-,18+,19+,20-,21-/m1/s1

7-(beta-D-Glucopyranosyloxy)-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one

Apigenin-7-O-β-D-glucopyranoside; Apigetrin; COSMOSIIN; Apigenin 7-glucoside; Cosmosiine; Cosmosioside; APIGENIN-7-GLUCOSIDE; Apigenin 7-O-beta-D-glucoside; ...; 578-74-5; . Cosmosiin; Apigetrin; COSMOSIIN; Apigenin 7-glucoside; Cosmosiine; Cosmosioside; APIGENIN-7-GLUCOSIDE; Apigenin 7-O-beta-D-glucoside; Apigenin 7-glucoside

2934.99.9001

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 : ≥ 100 mg/mL (~231.28 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.78 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 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: ≥ 2.5 mg/mL (5.78 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 25.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)