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

人羰基还原酶1（CBR1）是导致抗肿瘤药物蒽环类治疗效率降低的酶之一，在酿酒酵母中功能性表达。使用柔红霉素作为底物纯化CBR1并进行动力学表征。CBR1催化柔红霉素的还原遵循明显的米氏动力学，K（M）=85.2+-26.7microM，V（max）=3490+/-220micromol/（mingprotein）。通过研究Rutin/芦丁存在下的初始反应速率，确定了黄酮类化合物芦丁的抑制类型。发现抑制动力学遵循明显的混合抑制，K（ic）=1.8+/-1.2microM和K（iu）=2.8+/-1.6microM。还测定了一组黄酮类化合物的IC50值，以确定抑制活性的基本结构。四种最佳抑制剂与CBR1催化位点的计算对接实验表明，黄酮骨架结构是分子的结合部分。1和2中的糖部分，或9中的糖模拟部分的存在，引导了黄酮的方向，使糖向外指向，从而对结合产生稳定作用。最后，鉴定了与黄酮配体不同部分相互作用的其他结合表位，这些表位可能成为进一步提高抑制活性的靶点。其中包括：；Ser139和Cys226、Met234和Tyr193或Trp229周围的氢结合位点；与Tyr193、Trp229或NADPH的芳香-芳香相互作用；范德华与Ile140的相互作用[5]。

芦丁水合物可增加乙酰胆碱水平，抑制 JNK 和 ERK1/2 激活，并激活 mTOR 信号传导，从而改善氯化镉诱导的大鼠空间记忆丧失和神经元死亡 [4]。

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阿尔茨海默病（AD）是一种进行性神经退行性疾病，其特征是脑内细胞外β-淀粉样蛋白（aβ）斑块和细胞内神经原纤维缠结。Aβ聚集与神经毒性、氧化应激和神经元炎症密切相关。可溶性Aβ低聚物被认为是所有形式的Aβ聚集体中神经毒性最强的形式。我们之前报道过一种多酚化合物芦丁，它可以在体外抑制aβ聚集和细胞毒性，减轻氧化应激，减少一氧化氮和促炎细胞因子的产生。在目前的研究中，我们研究了芦丁对APPswe/PS1dE9转基因小鼠的影响。结果表明，口服芦丁显著减轻了AD转基因小鼠的记忆缺陷，降低了寡聚体Aβ水平，增加了超氧化物歧化酶（SOD）活性和谷胱甘肽（GSH）/谷胱甘肽二硫化物（GSSG）比值，降低了GSSG和丙二醛（MDA）水平，下调了小胶质细胞增生和星形胶质细胞增生，降低了脑中白细胞介素（IL）-1β和IL-6水平。这些结果表明，芦丁因其抗氧化、抗炎和降低aβ寡聚体活性而成为治疗AD的有前景的药物。[3]

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本研究旨在研究单独使用或与α-生育酚联合使用芦丁水合物（RH）对氯化镉（CdCl2）诱导的大鼠神经毒性和认知障碍的潜在神经保护作用，并探讨其作用机制。用赋形剂治疗CdCl2中毒的大鼠，检查RH、α-生育酚或联合治疗，并与接受赋形剂或单独剂量任一药物的对照组大鼠进行比较。数据证实，RH通过增加乙酰胆碱的可用性、提高内源性抗氧化能力、激活细胞存活和抑制凋亡途径来改善空间记忆功能，当RH与α-生育酚结合时，这种作用更有效。RH的作用机制包括激活PP2A介导的ERK1/2和JNK凋亡通路的抑制，以及抑制PTEN介导的mTOR存活通路的激活。总之，RH对CdCl2诱导的脑损伤和记忆功能障碍具有强大的神经保护作用，α-生育酚的联合给药可增强其活性[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

269.43

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.09 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (4.09 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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