Natural flavonoid; various bioactivity; Human carbonyl reductase 1 (CBR1)

Human carbonyl reductase 1 (CBR1), that is one of the enzymes responsible for the reduced efficiency of treatments by the antineoplastic agents anthracyclines, was functionally expressed in Saccharomyces cerevisiae. CBR1 was purified and kinetically characterised using daunorubicin as substrate. CBR1-catalysed reduction of daunorubicin followed an apparent Michaelis-Menten kinetics with K(M)=85.2+/-26.7microM and V(max)=3490+/-220micromol/(mingprotein). The type of inhibition for the flavonoid compound Rutin was determined by studying initial reaction rates in the presence of Rutin. The inhibition kinetics was found to follow an apparent mixed inhibition with K(ic)=1.8+/-1.2microM and K(iu)=2.8+/-1.6microM. IC50-values were also determined for a set of flavonoids in order to identify essential structure for inhibition activity. Computational docking experiments of the four best inhibitors to the catalytic site of CBR1 showed that the flavonoid skeleton structure was the binding part of the molecule. The presence of a sugar moiety in 1 and 2, or a sugar mimicking part in 9, directed the orientation of the flavonoid so that the sugars were pointing outwards, giving rise to a stabilising effect to the binding. Finally, additional binding epitopes that interacted with various parts of the flavonoid ligand were identified and could potentially be targeted for further improvement of inhibition activity. These included; hydrogen-binding sites surrounding Ser139 and Cys226, Met234 and Tyr193 or Trp229; aromatic-aromatic interaction with Tyr193, Trp229 or NADPH; van der Waals interactions with Ile140 [5].

Rutin hydrate increases acetylcholine levels, inhibits JNK and ERK1/2 activation, and activates mTOR signaling to ameliorate cadmium chloride-induced spatial memory loss and neuronal death in rats [4].

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Alzheimer's disease (AD) is a progressive, neurodegenerative disease characterized by extracellular β-amyloid (Aβ) plaques and intracellular neurofibrillary tangles in the brain. Aβ aggregation is closely associated with neurotoxicity, oxidative stress, and neuronal inflammation. The soluble Aβ oligomers are believed to be the most neurotoxic form among all forms of Aβ aggregates. We have previously reported a polyphenol compound Rutin that could inhibit Aβ aggregation and cytotoxicity, attenuate oxidative stress, and decrease the production of nitric oxide and proinflammatory cytokines in vitro. In the current study, we investigated the effect of rutin on APPswe/PS1dE9 transgenic mice. Results demonstrated that orally administered rutin significantly attenuated memory deficits in AD transgenic mice, decreased oligomeric Aβ level, increased super oxide dismutase (SOD) activity and glutathione (GSH)/glutathione disulfide (GSSG) ratio, reduced GSSG and malondialdehyde (MDA) levels, downregulated microgliosis and astrocytosis, and decreased interleukin (IL)-1β and IL-6 levels in the brain. These results indicated that rutin is a promising agent for AD treatment because of its antioxidant, anti-inflammatory, and reducing Aβ oligomer activities.[3]

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This study aimed at studying the potential neuroprotective effect of Rutin hydrate (RH) alone or in conjugation with α-tocopherol against cadmium chloride (CdCl2)-induced neurotoxicity and cognitive impairment in rats and to investigate the mechanisms of action. Rats intoxicated with CdCl2 were treated with the vehicle, RH, α-tocopherol or combined treatment were examined, and compared to control rats received vehicle or individual doses of either drug. Data confirmed that RH improves spatial memory function by increasing acetylcholine availability, boosting endogenous antioxidant potential, activating cell survival and inhibiting apoptotic pathways, an effect that is more effective when RH was conjugated with α-tocopherol. Mechanism of RH action includes activation of PP2A mediated inhibiting of ERK1/2 and JNK apoptotic pathways and inhibition of PTEN mediated activation of mTOR survival pathway. In conclusion, RH affords a potent neuroprotection against CdCl2-induced brain damage and memory dysfunction and co-administration of α-tocopherol enhances its activity [4].

Animal treatment [3]
APPswe/PS1dE9 transgenic mice expressing a chimeric mouse/human APP695 harboring the Swedish K670M/N671L mutations and human PS1 with the exon-9 deletion mutation were used for spatial memory test. The transgenic mice overproduce human Aβ40 and Aβ42 peptides. They also develop progressive cerebral β-amyloid deposit and learning and memory impairment. AD and WT littermate mice (males, 8 months of age) were given food and water ad libitum and kept in a colony room at 22 ± 2 °C and 45 ± 10% humidity under a 12:12 h light/dark cycle. All mice were separated into three groups: rutin-treated AD (n = 8), AD control (n = 8), and WT control (n = 8). The rutin-treated group was orally administered a daily dose of 100 mg/kg rutin for 6 wk. The mice in the AD control and WT groups were treated with 0.5% carboxymethylcellulose (CMC). Then, 5 d after the last administration, the mice were trained and tested in Morris water maze (MWM).

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MWM test [3]
The effect of rutin on the spatial cognitive performance of AD mice was investigated through the MWM test according to a previously described method. The mice were allowed to habituate for 1 wk and then tested in a water maze (1.1 m in diameter). The maze filled with water was drained daily. The temperature of the water was maintained at 22 ± 1 °C. The platform (10 cm in diameter) was fixed to 1 cm beneath the water surface throughout the training period, whereas the starting positions were counter balanced. All mice were initially assessed in the water maze to identify any inherent quadrant preference, and those exhibiting some preference were eliminated from subsequent testing. The mice were allowed to swim for 60 s to find the platform, on which they were allowed to stay for 10 s. Mice unable to locate the platform were guided to it. The mice were trained twice per day over five consecutive days, with an inter-trial interval of 3–4 h. The swimming activity of each mouse was monitored using a video camera mounted overhead and then automatically recorded via a video tracking system. At 24 h after the last learning trial, the mice were tested for memory retention in a probe trial without the platform.

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Rutin was dissolved in 0.01 g/ml CMC to a final concentration of 100 mg/ml. [4]
Experimental protocol [4]
Rats were divided into six groups (n = 10/group) and were treated as follows: [4]
(1) A control group: received of 0.01 g/ml carboxymethylcellulose (CMC) dissolved in distilled water; (2) α-tocopherol acetate treated group: control rats received α-tocopherol (120 IU/rat) diluted in 0.1 ml of coconut oil as previously described by Guimarães et al. (Citation2015) who have shown safe and highly neuroprotective effect of α-tocopherol acetate at this dose in a stroke animal model; (3) RH treated control group (control + RH): control rats received RH (100 mg/kg) as was studied by Vahideh et al. (Citation2014) who showed that RH is a safe and neuroprotective at this dose; (4) CdCl2 intoxicated group: received CdCl2 at a final dose of 5 mg/kg to induce neurotoxicity as shown by Shagirtha et al. (Citation2011); (5) CdCl2+RH treated group (CdCl2+RH): received CdCl2 (5 mg/kg) and received a coincided dose of RH (100 mg/kg body weight); (6) CdCl2+ RH + α-tocopherol acetate-treated group: received CdCl2 (5 mg/kg) and received concomitant dose of RH (100 mg/kg) in conjugation with α-tocopherol acetate (120 IU/rat) that is diluted in 0.1 ml of coconut oil. However, since no adverse neurological effects were seen when coconut oil was administered as a vehicle (Guimarães et al. Citation2015), we excluded this group of being a second control group in this study. All treatments were given by orogastric gavage with a polyethylene catheter PE 190 daily for 30 days.

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Assessment of cognitive performance [4]
Morris water maze (MWM) was used to assess the hippocampus-dependent spatial learning and memory function of all rats as previously described by Morris. The maze consists of a circular swimming pool (180 cm diameter) that is filled with water (50 cm deep, 20 ± 2 °C). The principle of the test is to determine the ability of the rats to remember and find an escape small circular platform (10 cm in diameter) that is submerged at 2 cm below the surface of the water over several various trails per day over four consecutive days. During the test, all rats were tested on an average of five trials/day (90 s/trial) for four consecutive days. In other words, all animals were allowed to swim for 90 s to find the hidden platform and each rat was allowed to remain on the platform for 30 s. If failed to find the platform, then it was manually guided to the platform. At the end of each trial, the average time of the five testing readings was presented as mean values of the cognitive performance.

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Brains collection and homogenates preparation [4]
Directly after the MWM test on day 34, all rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and their skulls were opened and the brains were quickly extracted on the ice, washed with cold phosphate buffer saline PBS and immediately placed in ice-cold dishes. The brain of each rat was cut into two halves longitudinally in which one-half was used to prepare homogenates (according to the manufacturer’s instruction) used in the biochemical determination and the other half was directly stored at −80 °C and used later for western blot study.

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Determination of the biochemical parameters in the brain homogenates [4]
Levels of reduced glutathione (GSH) and glutathione disulphide (GSSG) were determined using a rat’s colorimetric assay determination kit. Activities of superoxide dismutase (SOD) and glutathione peroxidase were determined using rat’s colorimetric assay determination kit. Levels of Malondialdehyde (MDA) were determined using a rat’s colorimetric determination kit (ab118970/Abcam, UK). Levels of acetylcholine (Ach), choline acetyltransferase (CAT) and acetylcholinesterase (AChE) were determined using rat’s special ELISA kits. All procedures were done in accordance with the manufacturer’s instructions.

5280805 Rat Intraperitoneal LD50 2 gm/kg Eksperimentalna Meditsina i Morfologiya., 19(207), 1980 [PMID:7460808]
5280805 Mouse Intraperitoneal LD50 200 mg/kg National Technical Information Service., AD277-689
5280805 Mouse Intravenous LD50 950 mg/kg Journal of the American Pharmaceutical Association, Scientific Edition., 39(556), 1950
5280805 Guinea Pig Intraperitoneal LD50 2 gm/kg Eksperimentalna Meditsina i Morfologiya., 19(207), 1980 [PMID:7460808]

[1]. Ghorbani A. Mechanisms of antidiabetic effects of flavonoid rutin. Biomed Pharmacother. 2017;96:305-312.

[2]. Habtemariam S. Rutin as a Natural Therapy for Alzheimer's Disease: Insights into its Mechanisms of Action. Curr Med Chem. 2016;23(9):860-873.

[3]. Rutin improves spatial memory in Alzheimer's disease transgenic mice by reducing Aβ oligomer level and attenuating oxidative stress and neuroinflammation. Behav Brain Res. 2014;264:173-180.

[4]. Rutin hydrate ameliorates cadmium chloride-induced spatial memory loss and neural apoptosis in rats by enhancing levels of acetylcholine, inhibiting JNK and ERK1/2 activation and activating mTOR signalling. Arch Physiol Biochem. 2018;124(4):367-377.

[5]. Flavonoids as inhibitors of human carbonyl reductase 1. Chem Biol Interact. 2008 Jul 30;174(2):98-108.

Rutin is a rutin glycoside formed by replacing the C-3 hydroxyl group of quercetin with glucose and rhamnose. It possesses metabolic and antioxidant properties. It is a disaccharide derivative belonging to the quercetin O-glucoside, tetrahydroxyflavone, and rutin glycosides. It is a flavonol glycoside found in various plants, including buckwheat, tobacco, forsythia, hydrangea, and violet. It has been used to treat capillary fragility. Rutin has also been reported in tea trees, amaranth, and other organisms with relevant data. Bioflavonoids are naturally occurring flavonoid or coumarin derivatives with so-called vitamin P activity, particularly rutin and aescin. It is a flavonol glycoside found in various plants, including buckwheat, tobacco, forsythia, hydrangea, and violet. It has been used to treat capillary fragility. See also: Quercetin (subclass); Ginkgo (part); Calendula (part)... See more... Multiple pieces of evidence suggest that flavonoids derived from vegetables and medicinal plants can have beneficial effects on diabetic patients by improving glycemic control, lipid profile, and antioxidant status. Rutin is a flavonoid found in many plants with a wide range of biological activities, including anti-inflammatory, antioxidant, neuroprotective, nephroprotective, and hepatoprotective effects. This article will explore the hypoglycemic properties of rutin and its protective effect against diabetic complications. The mechanisms by which rutin exerts its hypoglycemic effect may include: reducing the absorption of carbohydrates in the small intestine, inhibiting tissue gluconeogenesis, increasing tissue glucose uptake, stimulating insulin secretion from β-cells, and protecting pancreatic islets from degeneration. Rutin can also reduce the production of sorbitol, reactive oxygen species, precursors of advanced glycation end products (AGEs), and inflammatory cytokines. These effects are believed to be the reason why rutin has a protective effect against nephropathy, neuropathy, liver damage, and cardiovascular disease caused by hyperglycemia and dyslipidemia. In summary, current experimental findings support the potential of rutin in the prevention or treatment of diabetes-related diseases. Well-designed clinical studies are recommended to evaluate the advantages and limitations of rutin in diabetes management. [1]

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Rutin (quercetin-3-O-rutin glycoside) is a multifunctional natural flavonoid glycoside that has significant effects on a variety of cellular functions under pathological conditions. Rutin and/or its metabolites can cross the blood-brain barrier and have been shown to improve cognitive and various behavioral symptoms of neurodegenerative diseases. This review explores the potential of rutin in treating Alzheimer's disease (AD) by evaluating existing literature on various cellular and molecular targets associated with AD. Among the most relevant mechanisms are: influencing the processing, aggregation and action of β-amyloid (Aβ); altering the oxidative-antioxidant balance associated with neuronal cell loss; and removing inflammatory components of neurodegenerative diseases. Effects of rutin due to its physicochemical properties, such as metal chelation and bioavailability, are also discussed. [2]

C27H30O16

610.52

610.153

C, 53.12; H, 4.95; O, 41.93

153-18-4

Rutin hydrate;207671-50-9;Rutin trihydrate;250249-75-3

5280805

Light yellow to yellow solid powder

1.8±0.1 g/cm3

983.1±65.0 °C at 760 mmHg

195 °C (dec.)(lit.)

325.4±27.8 °C

0.0±0.3 mmHg at 25°C

1.765

1.76

10

16

6

43

1020

10

C[C@H]1[C@@H]([C@H]([C@H]([C@@H](O1)OC[C@@H]2[C@H]([C@@H]([C@H]([C@@H](O2)OC3=C(OC4=CC(=CC(=C4C3=O)O)O)C5=CC(=C(C=C5)O)O)O)O)O)O)O)O

IKGXIBQEEMLURG-NVPNHPEKSA-N

InChI=1S/C27H30O16/c1-8-17(32)20(35)22(37)26(40-8)39-7-15-18(33)21(36)23(38)27(42-15)43-25-19(34)16-13(31)5-10(28)6-14(16)41-24(25)9-2-3-11(29)12(30)4-9/h2-6,8,15,17-18,20-23,26-33,35-38H,7H2,1H3/t8-,15+,17-,18+,20+,21-,22+,23+,26+,27-/m0/s1

2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-[[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxymethyl]oxan-2-yl]oxychromen-4-one

Birutan; Rutin; 153-18-4; rutoside; Phytomelin; Birutan; Sophorin; Myrticolorin; Eldrin; Rutoside

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)

DMSO : ~50 mg/mL (~81.90 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.09 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 (4.09 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.)