Natural Polyphenols

In our continuing efforts to identify effective naturally sourced agents for diabetic complications, five caffeoylated phenylpropanoid glycosides, acteoside (1), isoacteoside (2), poliumoside (3), brandioside (4), and pheliposide (5) were isolated from the 80% EtOH extract of Brandisia hancei stems and leaves. These isolates (1-5) were subjected to an in vitro bioassay evaluating their inhibitory activity on advanced glycation end product formation and rat lens aldose reductase activity. All tested compounds exhibited significant inhibition of advanced glycation end product formation with IC50 values of 4.6-25.7 µM, compared with those of aminoguanidine (IC50=1,056 µM) and quercetin (IC50=28.4 µM) as positive controls. [1]

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Isoacteoside suppressed COX-2, iNOS, TNF-α, IL-6 and IL-1β expression. Furthermore, isoacteoside attenuated the LPS-induced transcriptional activity of NF-κB by decreasing the levels of phosphorylated IκB-α and IKK and NF-κB/p65 nuclear translocation. In addition, isoacteoside inhibited LPS-induced transcriptional activity of AP-1 by reducing the levels of phosphorylated JNK1/2 and p38MAPK. Isoacteoside blocked LPS-induced TLR4 dimerization, resulting in a reduction in the recruitment of MyD88 and TIR-domain-containing adapter-inducing interferon-β (TRIF) and the phosphorylation of TGF-β-activated kinase-1 (TAK1). [2]

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soacteoside exerted both dose-dependent as well as time-dependent growth inhibitory effects on ovarian cancer cells with IC50 values of 15 µM at 24h incubation. Isoacteoside led to early and late apoptosis induction in these cells. Isoacteoside also led to sub-G1 cell cycle arrest which showed strong dose-dependence. Isoacteoside treatment also led to inhibition of cell migration and cell invasion. [3]

In the rat lens aldose reductase assay, acteoside, isoacteoside, and poliumoside exhibited greater inhibitory effects on rat lens aldose reductase with IC50 values of 0.83, 0.83, and 0.85 µM, respectively, than those of the positive controls, 3,3-tetramethyleneglutaric acid (IC50=4.03 µM) and quercetin (IC50=7.2 µM). In addition, the effect of acteoside on the dilation of hyaloid-retinal vessels induced by high glucose in larval zebrafish was investigated. Acteoside reduced the diameters of high glucose-induced hyaloid-retinal vessels by 69% at 10 µM and 81% at 20 µM, compared to the high glucose-treated control group. These results suggest that B. hancei and its active components might be beneficial in the treatment and prevention of diabetic vascular complications. [1]

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Pretreatment of mice with isoacteoside effectively inhibited xylene-induced ear oedema and LPS-induced endotoxic death and protected against LPS-induced AKI. Conclusions and implications: Isoacteoside blocked TLR4 dimerization, which activates the MyD88-TAK1-NF-κB/MAPK signalling cascades and TRIF pathway. Our data indicate that isoacteoside is a potential lead compound for the treatment of inflammatory diseases. [2]

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The results revealed that OVCAR-3 tumor growth was significantly suppressed by isoacteoside administration, compared with that in the control group. At the end of the 5-week period of isoacteoside treatment, the average tumor growth and volume in the untreated control group were considerably higher than those in the treated groups. Conclusion: In brief, the current study indicates that isoacteoside has a great potential in suppressing both in vitro and in vivo ovarian cancer cell growth and can be used as a possible anticancer agent. [3]

Docking of isoacteoside to TLR4 complex [2]
A docking simulation of isoacteoside with the TLR4 complex (PDB code: 3FXI) was performed using the molecular modelling packages in Ledock (http://www.lephar.com). TLR4 complex crystal structures were obtained from the RCSB Protein Data Bank (PDB code: 3FXI) (Park et al., 2009).

MTT assay [2]
RAW264.7 cells were seeded into 96‐well plates at a density of 105 cells per well overnight. Then, the cells were treated with isoacteoside (20, 40 or 80 μM) for 24 h, and the MTT assay was used to determine the cytotoxicity according to our previous report (Gao et al., 2015).

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Determination of nitrite and NO [2]
RAW264.7 cells were plated in 24‐well plates at a density of 5 × 105 per well overnight. After the cells were pretreated with isoacteoside (20, 40 or 80 μM) for 1 h, LPS (1 μg·mL−1) was added to the isoacteoside containing medium and cultured for 24 h. The nitrite levels in the culture media were determined using the Griess reagent according to the protocol from the manufacturer. Treated cells were collected and stained with DAF‐FM (1 μM) for an additional 1 h. The fluorescence signal was determined by FACScan flow cytometry using the FITC channel.

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ELISA [2]
After being pretreated with isoacteoside (20, 40 or 80 μM) for 1 h, BMDMs and RAW264.7 cells were then incubated with LPS (1 μg·mL−1) for 24 h. The medium was collected, and the secretion levels of TNF‐α, IL‐6 and IL‐1β were determined using ELISA kits according to the manufacturer's instructions.

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Immunofluorescence [2]
The immunofluorescence analysis of NF‐κB/p65 was conducted as previously described (Gao et al., 2016). Briefly, 2 × 105 RAW264.7 cells were seeded into a 35‐mm glass bottom SPL confocal dish overnight. After the cells were pretreated with isoacteoside (80 μM) for 1 h and stimulated with LPS (1 μg·mL−1) for another 2 h, the primary antibody anti‐NF‐κB/p65 (1:50) and secondary antibody goat anti‐rabbit Alexa Fluor 568 (1:200) were applied. Cells were fixed and stained with Hoechst 33 342 (1 μM) for imaging. The imaging was performed under a Leica TCS SP8 laser confocal microscope at 60× magnification using excitation/emission wavelengths of 588/615–690 nm.

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Transient transfection and luciferase assay [2]
HEK293T cells were seeded into a dish (10 cm, i.d.), at a density of 106 cells, and left overnight. TLR4‐HA and TLR4‐Flag plasmids obtained from Addgene were co‐transfected using TurboFect transfection reagents for 24 h. The transfected cells were seeded into a dish (5 cm, i.d.) at a density of 5 × 105 cells, and left overnight. The cells were pretreated with isoacteoside (80 μM) for 1 h and stimulated with LPS (1 μg·mL−1) for 24 h before harvesting.
RAW264.7 cells cultured in 96‐well plates overnight were transiently co‐transfected with piNOS‐luc, pNF‐κB‐luc or pAP‐1‐luc plasmids and pRL‐TK plasmid to detect iNOS, NF‐κB or AP‐1 transcriptional activity, respectively, according to the manufacturer's instructions. The pRL‐TK plasmid was used as the reference control. After 48 h of transfection, the cells were pretreated with isoacteoside (80 μM) for 1 h and stimulated with LPS (1 μg·mL−1) for 16 h. The luciferase activity was determined using a Dual‐Glo luciferase assay system kit according to the manufacturer's instructions.

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The expression of the NF-κB pathway, MAPK pathway, iNOS, TNF-α, IL-6 and IL-1β was evaluated using Western blotting, quantitative real-time PCR or ELISA. TLR4 dimerization was determined by transfecting HEK293T cells with TLR4 plasmids. [2]

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MTT assay was used to study the cytotoxic effects of the compound on the cell viability. Effects on apoptosis and cell cycle arrest were evaluated by flow cytometry. In vitro wound healing assay and matrigel assay were carried out to study the effects of isoacteoside on cell migration and cell invasion respectively. Non-cancer ovarian cell line SV-40 served as control.[3]

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MTT assay [3]
The proliferation of the ovarian cancer and normal cells was assessed by MTT assay. In brief, the cells were cultured in 96-well flat-bottom microtiter culture plates at a density of 5×104 cells per well and subjected to treatment with isoacteoside at varied concentrations for 24 hrs. Afterwards, the addition of fresh MTT solution followed, and then the cells were incubated for another 4 hrs at 37°C. This was followed by addition of DMSO to dissolve the formazan crystals. Finally, the absorbance was taken at 570 nm using a microplate reader.

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Matrigel invasion assay [3]
Invasion was evaluated with the help of Matrigel®- coated invasion chambers. The isoacteoside-treated and untreated OVCAR-3 cells that reached the lower surface of the membrane were subjected to staining with crystal violet (CV), and images of CV-stained cells were taken. The crystal violet complexes formed were dissolved in 10% acetic acid and the cell invasion was determined by measuring the absorbances of the resultant solutions at 600 nm in a spectrophotometer.

The in vivo anti-inflammatory effect of isoacteoside was determined using mouse models of xylene-induced ear oedema, LPS-induced endotoxic shock and LPS-induced endotoxaemia-associated acute kidney injury (AKI). [2]

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Animal experiments and ethical statement BALB/c mice (male, 6–8 weeks‐old and weighing 18–22 g) were reared in plastic cages (≤5 mice per cage) and given free access to food and water under standard conditions (specific‐pathogen‐free) with air filtration (22 ± 2°C, 12‐h light/12‐h dark).
For the xylene‐induced mice ear oedema model (Ma et al., 2013), isoacteoside (100 mg·kg−1) was administered i.p. After 2 h, 30 μL xylene (≥98.5%) was applied to the posterior and anterior surfaces of the right ears. The left ears were untreated and served as controls. An hour later, the mice were killed by cervical dislocation under ether anaesthesia, and two ear punches (7 mm, i.d.) were collected and weighed. The oedema was evaluated by comparing the increase in the weight of the right ear punch with the increase in the weight of the left ear punch. The ear tissues were collected and fixed in 10% formaldehyde for at least 24 h at room temperature. After being dehydrated in different concentrations of alcohol, tissues were embedded in paraffin and sliced. The sections were stained with haematoxylin and eosin (H&E) and imaged under a light microscope.
For the endotoxic shock model (Chen et al., 2015b), endotoxaemia was induced in mice by administering LPS (20 mg·kg−1, i.p.). The mice were pretreated with isoacteoside (100 mg·kg−1, i.p.) for 2 h before the LPS injection. Dexamethasone (DEX, 5 mg·kg−1, i.p.) was used as positive control because it has a superior anti‐inflammatory effect (Zeng et al., 2015; Qin et al., 2016) and has been used in a clinical study to treat an endotoxic shock (Zhang et al., 2003). The survival rate was monitored continuously for 132 h. After 132 h, the remaining mice were killed by cervical dislocation under ether anaesthesia.
For the AKI model (Zarjou and Agarwal, 2011), mice were randomly divided into six groups: a control group, isoacteoside (25, 50 and 100 mg·kg−1, i.p.) groups, an LPS group (10 mg·kg−1, i.p.) and a positive control dexamethasone (5 mg·kg−1, i.p.) group. At 2 h before the LPS injection, mice were pretreated with isoacteoside. After 12 h, serum samples were collected by retro‐orbital bleeding under ether anaesthesia, and the cytokines (TNF‐α, IL‐6 and IL‐1β) were examined using ELISA kits according to the manufacturer's instructions. The mice were killed by cervical dislocation. The serum levels of urea nitrogen (BUN) and creatinine were determined on a Roche Module P800. Furthermore, the kidney tissues were harvested and fixed in 10% formaldehyde. After being dehydrated in different concentrations of alcohol, the tissues were embedded in paraffin and sliced. The sections were stained with H&E and imaged under a light microscope. [2]

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> In vivo study [3]
In this study 4 week-old female mice were injected with 5×106 OVCAR-3 cells subcutaneously at the left flank. The mice were then randomly divided into two groups. As the tumors became apparent, one group of mice (n=5) was injected intraperitoneally with DMSO (0.1%) dissolved isoacteoside and diluted with 100 μL normal saline at 30 mg/kg body weight and this time point was taken as the first day of the experiment. isoacteoside was administered to the mice thrice a week, while the control mice were administered DMSO (0.1%) in normal saline only. At the end of 5 weeks, the mice were sacrificed and tumors were harvested for assessment of tumor growth and other investigations.

[1]. Caffeoylated phenylpropanoid glycosides from Brandisia hancei inhibit advanced glycation end product formation and aldose reductase in vitro and vessel dilation in larval zebrafish in vivo. Planta Med. 2013 Dec;79(18):1705-9.

[2]. Isoacteoside, a dihydroxyphenylethyl glycoside, exhibits anti-inflammatory effects through blocking toll-like receptor 4 dimerization. Br J Pharmacol. 2017;174(17):2880-2896.

[3]. Yang X, Guo F, Peng Q, Liu Y, Yang B. Suppression of in vitro and in vivo human ovarian cancer growth by isoacteoside is mediated via sub-G1 cell cycle arrest, ROS generation, and modulation of AKT/PI3K/m-TOR signalling pathway. J BUON. 2019;24(1):285-290.

Isoacteoside is a hydroxycinnamic acid.
Isoacteoside has been reported in Acanthus ebracteatus, Magnolia officinalis, and other organisms with data available.
See also: Harpagophytum zeyheri root (part of).

C29H36O15

624.5872

624.205

61303-13-7

6476333

White to yellow solid powder

1.6±0.1 g/cm3

942.9±65.0 °C at 760 mmHg

220-230℃

306.1±27.8 °C

0.0±0.3 mmHg at 25°C

1.689

2.58

9

15

11

44

936

10

O([C@@]1([H])[C@@]([H])([C@@]([H])([C@]([H])([C@]([H])(C([H])([H])[H])O1)O[H])O[H])O[H])[C@]1([H])[C@]([H])([C@]([H])(OC([H])([H])C([H])([H])C2C([H])=C([H])C(=C(C=2[H])O[H])O[H])O[C@]([H])(C([H])([H])OC(/C(/[H])=C(\[H])/C2C([H])=C([H])C(=C(C=2[H])O[H])O[H])=O)[C@@]1([H])O[H])O[H]

FNMHEHXNBNCPCI-QEOJJFGVSA-N

InChI=1S/C29H36O15/c1-13-22(35)24(37)25(38)29(42-13)44-27-23(36)20(12-41-21(34)7-4-14-2-5-16(30)18(32)10-14)43-28(26(27)39)40-9-8-15-3-6-17(31)19(33)11-15/h2-7,10-11,13,20,22-33,35-39H,8-9,12H2,1H3/b7-4+/t13-,20+,22-,23+,24+,25+,26+,27-,28+,29-/m0/s1

[(2R,3R,4S,5R,6R)-6-[2-(3,4-dihydroxyphenyl)ethoxy]-3,5-dihydroxy-4-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-2-yl]methyl (E)-3-(3,4-dihydroxyphenyl)prop-2-enoate

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 : ~250 mg/mL (~400.26 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.33 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 (3.33 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 (3.33 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.)