Natural flavonoid in green tea; COX-1/cyclooxygenase-1

At 70 μg/mL and an IC50 of 1.4 μM, (+)-catechin demonstrates >95% inhibitory action against cyclooxygenase-1 (COX-1) [1]. Following a 24-hour treatment with (+)-catechin, a dose-dependent decrease in color was noted. At the highest concentration of (+)-catechin tested (160 μg/mL), 54.76% of cells perished, with an IC50 of (+)-catechin at 127.62 μg/mL. Treatment of MCF-7 cells with (+)-catechin resulted in an increase in the induction of apoptosis that was dose- and time-dependent. After 24 hours, 40.7% and 41.16% of the cells treated with 150 μg/mL and 300 μg/mL (+)-catechin experienced apoptosis, in comparison to the control cells. Following a 24-hour treatment with 150 μg/mL (+)-catechin, MCF-7 cells exhibited 5.81, 1.42, 3.29, and 2.68-fold increases in Caspase-3, -8, and -9 expression levels, respectively. Comparison with untreated control cell levels [2].

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(+)-Catechin Hydrate (31.23–250 μg/mL) was safe in undifferentiated IMR-32 neuroblastoma cells with an IC50 of 821.1 μg/mL. Co-treatment with catechin (31.23–250 μg/mL) dose-dependently protected against doxorubicin (1 or 2 μg/mL)-induced cell death; the IC50 of catechin in the presence of doxorubicin (1 μg/mL) was 37.61 μg/mL, and with doxorubicin (2 μg/mL) it was 42.87 μg/mL. In differentiated IMR-32 cells, doxorubicin (1.5 μg/mL) significantly reduced neurite length, and pretreatment with catechin (40 μg/mL) significantly increased neurite length compared to doxorubicin alone. Cell cycle analysis showed that doxorubicin (1.5 μg/mL) caused arrest at S and G2/M phases; catechin (40 μg/mL) pretreatment partially prevented this cell cycle arrest. [1]

Although the difference was not statistically significant, animals treated with (+)-catechin at the lowest tested dose (i.e., 50 mg/kg, po) explored unfamiliar targets substantially more frequently in choice trials. Time-induced episodic memory losses are prevented by (+)-catechin in a dose-dependent manner; 200 mg/kg orally is the most efficacious dose. Treatment with (+)-catechin prevented an increase in MPO levels (21.98±9.44% in the hippocampus and 36.76±4.39% in the frontal cortex) compared to the DOX treatment group alone [3].

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In time-induced episodic memory deficit model (24 h inter-trial interval) in Wistar rats, oral administration of (+)-Catechin Hydrate at 50, 100, and 200 mg/kg for 7 days prior to and during trials dose-dependently reversed memory deficits, as shown by increased novel object exploration time, recognition index, and discrimination index; the most effective dose was 200 mg/kg. In doxorubicin-induced memory deficit model (2.5 mg/kg i.p., 10 cycles over 50 days), daily oral catechin at 100 mg/kg for 57 days (including 7 days pre-treatment) prevented episodic memory deficits, with significantly increased recognition and discrimination indices compared to doxorubicin alone. Catechin treatment also significantly decreased doxorubicin-induced oxidative stress markers (reduced lipid peroxidation, increased catalase, SOD, and GSH levels) in hippocampus and frontal cortex, reduced nitrite levels and myeloperoxidase (MPO) activity (percentage increase in MPO: from 62.09% to 21.98% in hippocampus, and from 55.48% to 36.76% in frontal cortex), and reduced acetylcholinesterase activity in hippocampus (from 0.020 ± 0.002 to 0.016 ± 0.001 μmol/min/mg protein). Body weight: doxorubicin significantly reduced body weight compared to control (205.5 ± 8.35 g vs 247.83 ± 5.23 g on day 50); catechin co-treatment did not show significant additional weight loss (194.38 ± 7.55 g). [1]

Ex vivo acetylcholinesterase (AChE) inhibition assay: rat brain homogenate was incubated with different concentrations (500 to 1.25 μg/mL) of (+)-Catechin Hydrate for 45 minutes, and AChE activity was measured by Ellman’s method (using acetylthiocholine iodide and DTNB). The IC50 for catechin was 21.15 μg/mL (compared to donepezil IC50 = 79.11 μg/mL). [1]

Natural flavonoid in green tea In vitro studies[2]
IMR-32 cell line is male Caucasian derived neuroblastoma cell line. It was procured from NCCS, subcultured in DMEM medium with 10% of fetal bovine serum. The cells were used for cell viability and cyto-protection studies. The treatments of Catechin and DOX in the neuroprotection study were simultaneous (without changing the medium), where Catechin was added 1 h prior to DOX addition.

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MTT assay in undifferentiated IMR-32 cells[2]
Five thousand cells per well were seeded in microplate consisting of 50 µl of medium and incubated for 24 h. After 24 h, 50 µl of Catechin was added in a concentration ranging from 31.23 to 250 µg/ml in the wells for one hour. After that 50 µl of DOX (1 or 2 µg/ml) was added and incubated for 24 h. Followed by that 50 µl of MTT (2 mg/ml) was added and incubated at 37 °C for 3 h, after which the medium was removed and 100 µl of DMSO was added, and shaken for about 5 min on an orbital shaker. Formazan crystals formed were allowed to dissolve in DMSO. The absorbance of DMSO solubilized formazan was read at 540 nm (Shi et al. 2015). IC50 of Catechin was calculated by fitting the data to non-linear regression using GraphPad Prism.

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Cell cycle analysis[2]
Flow cytometric technique was used to evaluate the effect of Catechin on DOX- induced alteration of cell cycle. One million differentiated cells were seeded in flasks for overnight and incubated with Catechin (40 µg/ml) at 37 °C for 2 h, followed by DOX at 1.5 µg/ml for next 24 h. Cells were separated from the flask by trypsinization, washed with PBS with centrifugation. The cell pellets were collected and fixed with 70% ice-cold methanol and stored for 24 h at −20 °C. Then cell pellets were washed with PBS and added isotonic PI solution (25 µM propidium iodide, 0.03% NP-40 and 40 µg/ml RNase A) for staining. The stained cells were analyzed with Accuri C6 flow cytometer for cell cycle study at excitation wavelength 488 nm and emission wavelength 575/40 nm (Reddy et al. 2015; Simon et al. 2016).

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MTT assay in undifferentiated IMR-32 cells: 5,000 cells/well were seeded in 96-well plates. After 24 h, catechin (31.23–250 μg/mL) was added for 1 h, then doxorubicin (1 or 2 μg/mL) was added and incubated for 24 h. MTT (2 mg/mL) was added for 3 h at 37°C, then medium removed, DMSO added, and absorbance read at 540 nm. [1]
Neurite length assay in differentiated IMR-32 cells: Cells were differentiated using 5% FBS and 10 μM all-trans-retinoic acid for 10 days. On day 13, cells were treated with catechin (40 μg/mL) for 1 h, then doxorubicin (1.5 μg/mL) for 48 h. Neurite length was measured using ImageJ software under inverted microscope (40× objective); defined as straight-line distance from neurite tip to cell body junction. Approximately 100 images per treatment well were analyzed. [1]
Cell cycle analysis: Differentiated IMR-32 cells (1 million) were treated with catechin (40 μg/mL) for 2 h, then doxorubicin (1.5 μg/mL) for 24 h. Cells were trypsinized, fixed with 70% ice-cold methanol at -20°C for 24 h, washed, stained with propidium iodide solution (25 μM PI, 0.03% NP-40, 40 μg/mL RNase A), and analyzed by flow cytometry at excitation 488 nm, emission 575/40 nm. [1]

In vivo studies[2]
Selection of doses In the preliminary experiments for assessing the procognitive effect of Catechin, the selected doses were 50, 100 and 200 mg/kg for dose–response analysis. In later studies for chemobrain i.e., DOX-induced memory deficit model, the dose of Catechin selected was 100 mg/kg as it showed a promising effect in preliminary studies and moreover, the treatment was on a chronic basis. The dose of DOX selected was 2.5 mg/kg according to the previous studies and standardized laboratory procedures (Steiniger et al. 2004; Swamy et al. 2011; Grandhi et al. 2016).[2]
Preparation and administration of drugs In the preliminary study for assessing the nootropic effect of Catechin using time induced memory deficit model, the doses were prepared at 50, 100, 200 mg/kg in 0.25% w/v sodium carboxy methylcellulose (CMC) and administered orally for 7 days prior to and during the experimental trials. Four experimental groups were used (n = 9 each) for one vehicle (CMC) and three groups of Catechin (three doses).[2]
For inducing neurotoxicity and systemic toxicity, DOX (Adriamycin at 2.5 mg/kg) was administered intraperitoneally in 10 cycles on every 5 days. Three experimental groups (n = 12 each) were used viz., vehicle control (0.25% w/v CMC), DOX alone and Catechin (100 mg/kg in 0.25% CMC p.o.). Catechin was administered orally for 57 days including one-week treatment prior to the first cycle of DOX. Following the last cycle of DOX on day 57, i.e., on day 58, the behavioral study was conducted. All treatments, as well as the behavioral analysis, were carried out between 9 a.m. to 4 p.m. Body weight of the animals was taken once in 3 days throughout the study. During the experimental trials, the oral treatment was given 1 h before the familiarization trial.

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Time-induced natural memory deficits model[2]
Episodic memory deficits can be induced in rats naturally by increasing the time delay between familiarization and choice trials. Hence the initial experiment was conducted to assess the effect of Catechin on time -induced memory deficits by using an ITI of 24 h. In this test, rats were habituated to the arenas on day 1 and were subjected to familiarization trial on day 2. Then after an ITI of 24 h, i.e., on day 3, animals were subjected to recognition trial with one novel object replacing the familiar object. Four experimental groups were used. Rats were treated with either Catechin (50, 100 and 200 mg/kg, p.o.) or CMC (10 ml/kg, p.o.) for 7 days before the trial initiation. During the experimental trials, treatment was given 1 h before the actual trial in familiarization and choice trials

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Time-induced memory deficit model: Male Wistar rats (200–230 g) were orally administered (+)-Catechin Hydrate at 50, 100, or 200 mg/kg in 0.25% w/v sodium carboxymethylcellulose (CMC) for 7 days before and during trials. Novel object recognition test (NORT) was performed with a 24 h inter-trial interval. Treatment was given 1 h before familiarization and choice trials. [1]
Doxorubicin-induced memory deficit model: Rats received doxorubicin 2.5 mg/kg intraperitoneally every 5 days for 10 cycles (total 50 days). Catechin (100 mg/kg in 0.25% CMC) was given orally daily for 57 days (starting 7 days before first doxorubicin cycle). NORT was performed on day 58 with a 4 h inter-trial interval. After behavioral testing, rats were sacrificed under ketamine anesthesia; blood was collected via retro-orbital puncture, and brains (hippocampus and frontal cortex) were isolated for biochemical assays. [1]

In the doxorubicin-induced toxicity model, (+)-Catechin Hydrate at 100 mg/kg orally for 57 days did not cause significant additional weight loss compared to doxorubicin alone (doxorubicin+catechin: 194.38 ± 7.55 g vs doxorubicin alone: 205.5 ± 8.35 g).[1]

[1]. Potential cancer-chemopreventive activities of wine stilbenoids and flavans extracted from grape (Vitis vinifera) cell cultures. Nutr Cancer. 2001;40(2):173-9.

[2]. Catechin ameliorates doxorubicin-induced neuronal cytotoxicity in in vitro and episodic memory deficit in in vivo in Wistar rats. Cytotechnology. 2018 Feb;70(1):245-259.

[3]. Alshatwi AA. Catechin hydrate suppresses MCF-7 proliferation through TP53/Caspase-mediated apoptosis. J Exp Clin Cancer Res. 2010 Dec 17;29:167.

(+)-Catechin monohydrate is the monohydrate of (+)-catechin. It has anti-aging effects. It contains (+)-catechin. (+)-Catechin is the (+)-enantiomer of catechin and is a polyphenolic antioxidant plant metabolite. It has antioxidant and plant metabolic effects. It is the enantiomer of (-)-catechin. It is an antioxidant flavonoid compound, mainly found in woody plants, existing in both (+)-catechin and (-)-epicatechin (cis) forms. Anthocyanin alcohols have been reported to be found in tea (Camellia sinensis), peony (Paeonia obovata), and other organisms with relevant data. Catechins are metabolites found or produced in Saccharomyces cerevisiae.
An antioxidant flavonoid compound, mainly found in woody plants, exists in two forms: (+)-catechin and (-)-epicatechin (cis).
See also: gallocatechin (with subclasses); Croferlemer (monomer); blueberry (partial)...

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Moderate wine consumption is associated with a reduced risk of cancer. Twelve phenolic compounds were purified using grapevine cell culture: stilbene compounds trans-astraline, trans-sprucetin (2), trans-resveratrol, trans-resveratrol, trans-sprucetanol, cis-resveratrol, cis-sprucetin, and cis-resveratrol; flavane compounds (+)-catechin, (-)-epicatechin, and epicatechin 3-O-gallate; and flavane dimer proanthocyanidin B2 3'-O-gallate. In organ culture, the ability of these compounds to inhibit cyclooxygenase and precancerous lesion formation was evaluated using carcinogen-treated mouse mammary glands. At a concentration of 10 μg/ml, trans-astrigin and trans-picetol inhibited the development of 7,12-dimethylbenzo[a]anthracene-induced mouse breast mammary lesions, with inhibition rates of 68.8% and 76.9%, respectively (compared to untreated mammary glands). Of the 12 compounds tested in this study, trans-resveratrol exhibited the strongest activity, except for trans-resveratrol (87.5% inhibition). In cyclooxygenase (COX)-1 activity assays, the trans isomers of stilbene compounds appeared to be more active than the cis isomers: the half-maximal inhibitory concentration (IC50) of trans-resveratrol was 14.9 μM (96% inhibition), while the IC50 of cis-resveratrol was 55.4 μM. In the COX-2 activity assay, only trans- and cis-resveratrol showed significant inhibitory activity among the compounds tested (IC50 values of 32.2 and 50.2 μM, respectively). This is the first report of potential cancer chemopreventive activity for trans-astrinin, a plant stilbene compound recently discovered in wine. Trans-astrinin and its aglycone trans-piperidine were active in mouse mammary organ culture assays but showed no activity in the COX-1 and COX-2 activity assays. Trans-resveratrol was active in all three bioassays used in this study. These findings suggest that trans-astrinin and trans-piperidine may exert potential cancer chemopreventive effects through mechanisms different from those of trans-resveratrol. [1]

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Cognitive impairment caused by chemotherapy can impair the quality of life of cancer patients. Tea polyphenols are known chemopreventive agents. This study aimed to evaluate the neuroprotective effects of tea polyphenol(+) catechin hydrate (catechin) on IMR-32 neuroblastoma cells in vitro and its alleviating effect on episodic memory impairment in Wistar rats in vivo (induced by the commonly used chemotherapy drug doxorubicin (DOX)). In in vitro experiments, we assessed the neuroprotective effect on undifferentiated IMR-32 cells using cell viability and the neuroprotective effect on differentiated cells using neurite length. The results showed that catechin increased the viability of undifferentiated IMR-32 cells. Catechin pretreatment also increased the neurite length of differentiated cells. In in vivo experiments, we used a time-induced memory impairment model (dose of 50, 100, and 200 mg/kg) and a DOX-induced memory impairment model (dose of 100 mg/kg) to assess the neuroprotective effects of catechin through a novel object recognition task. The latter model was established by intraperitoneal injection of doxorubicin (DOX, 2.5 mg/kg) in Wistar rats for 50 consecutive days in 10 cycles. Catechins significantly reversed time-induced memory deficits in a dose-dependent manner and prevented DOX-induced memory deficits at a dose of 100 mg/kg. Furthermore, in a DOX-induced toxicity model, catechin treatment significantly reduced oxidative stress, acetylcholinesterase activity, and neuroinflammation in the hippocampus and cerebral cortex. Therefore, catechins may be a potential adjunctive therapy for improving DOX-induced cognitive impairment, thereby improving the quality of life of cancer survivors. This improvement may be attributed to enhanced antioxidant defense, prevention of neuroinflammation, and inhibition of acetylcholinesterase. [2] Catechin hydrate (CH) is a potent antioxidant capable of scavenging free radicals. It is a phenolic compound extracted from plants and found in natural foods and beverages such as green tea and red wine. CH has anticancer potential. The mechanism of action of many anticancer drugs is based on their ability to induce apoptosis. In this study, I aimed to identify downstream apoptosis genes targeted by CH in MCF-7 human breast cancer cells. CH effectively killed MCF-7 cells by inducing apoptosis. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and real-time PCR confirmed the occurrence of apoptosis. After 24 hours of exposure to 150 μg/ml and 300 μg/ml CH, the proportion of apoptotic cells was 40.7% and 41.16%, respectively. In addition, after 48 hours of exposure to 150 μg/ml and 300 μg/ml CH, the proportion of apoptotic cells was 43.73% and 52.95%, respectively. Interestingly, after 72 hours of exposure to both concentrations of CH, almost 100% of the cells lost their integrity. Real-time quantitative PCR further confirmed these results, namely that the expression of caspase-3, -8 and -9 and TP53 increased in a time- and dose-dependent manner. In summary, the ability of CH to induce apoptosis is related to its ability to increase the expression of pro-apoptotic genes such as caspase-3, -8 and -9 and TP53. [3]

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(+)-Catechin Hydrate is a tea polyphenol (flavan-3-ol) with antioxidant, anti-inflammatory, and neuroprotective properties. In this study, it ameliorated doxorubicin-induced neuronal cytotoxicity in vitro and episodic memory deficit in vivo, possibly via elevation of antioxidant defense, prevention of neuroinflammation, and inhibition of acetylcholinesterase. The study suggests catechin may be a potential adjuvant therapy for chemotherapy-induced cognitive impairment (“chemobrain”). [1]

C15H14O6

290.2681

308.089

C, 58.44; H, 5.23; O, 36.33

225937-10-0

(±)-Catechin;7295-85-4;Catechin;154-23-4

107957

Off-white to yellow solid powder

630.4ºC at760mmHg

175-177ºC

335ºC

9.29E-17mmHg at 25°C

1.481

6

7

1

22

364

2

C1[C@@H]([C@H](OC2=CC(=CC(=C21)O)O)C3=CC(=C(C=C3)O)O)O.O

OFUMQWOJBVNKLR-NQQJLSKUSA-N

InChI=1S/C15H14O6.H2O/c16-8-4-11(18)9-6-13(20)15(21-14(9)5-8)7-1-2-10(17)12(19)3-7;/h1-5,13,15-20H,6H2;1H2/t13-,15+;/m0./s1

(2R,3S)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol;hydrate

(+)-Catechin Hydrate; 225937-10-0; Catechin hydrate; 88191-48-4; (+)-catechin monohydrate; (2R,3S)-2-(3,4-Dihydroxyphenyl)chroman-3,5,7-triol hydrate; MFCD00149354; (+)-Cyanidol-3;

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (Infinity 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 (Infinity 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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Solubility in Formulation 3: ≥ 2.5 mg/mL (Infinity 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 25.0 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.)