Endogenous metabolite; glucuronidated metabolite of Baicalein; Natural flavone

• Baicalein was found to be abundant in the intestine after oral administration.[1]
• The amount of baicalein can support its intestinal pharmacological activities.[1]
• Baicalein metabolites were of different compositions in different intestinal tissues.[1]
• Phase II metabolites of baicalein were the major metabolites in the small intestine.[1]
• Metabolites produced by gut microbiota were of high abundance in the large intestine.[1]

In vitro metabolism of baicalein by fecal bacteria[1]
Fresh mice feces were thoroughly suspended in the GAM broth (10%, w/v) under anaerobic environment and then cultured in an anaerobic incubator at 37 °C for 24 h. The culture obtained was then used as mice intestinal bacterial mixture (Xu, Zhao, et al., 2014). Flavonoids were dissolved in DMSO and were added to 5 mL of mice intestinal bacterial mixture. The final concentrations of baicalein and DMSO were 0.74 mM and 2.5% (v/v), respectively. The mixtures containing flavonoids and blank GAM broth were set as control. Then the mixture was anaerobically incubated at 37 °C for 24 h. A total of 400 μL of acetonitrile/acetic acid (9:1, v/v), containing 74 μM of IS was added to 100 μL of the mixtures. After being centrifuged at 13000g for 10 min, the supernatant was collected and analyzed by HPLC. A culture-independent method was used to confirm the result. The fecal microbiota was prepared following the method published previously (Xia et al., 2012). Briefly, 1 g of fresh feces was collected and suspended in 9 mL of HBSS buffer at 4 °C under anaerobic environment. The suspension was centrifuged at 500g for 5 min. The supernatant was used as gut microbiota mixture. The subsequent procedures were carried out following the aforementioned method.

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In vitro metabolism of baicalein by mice intestine and liver S9 fractions[1]
The preparation of mice intestinal and hepatic S9 fractions was performed according to the method described previously (Ge et al., 2018, Xia et al., 2012). Baicalein (final concentration, 300 μM) was pre-incubated with mice liver or intestine S9 fraction (0.4 mg/mL and 2.5 mg/mL for glucuronidation and methylation, respectively) in Tris-HCl (0.1 M, pH 7.4) containing 8 mM MgCl2, 25 μg/mL alamethicin, and 0.4 mM saccharolactone for glucuronidation; and in Tris-HCl (0.1 M, pH 7.4) containing MgCl2 (8 mM) for methylation (Fong et al., 2012, Zhang et al., 2011). The reaction was initiated by the addition of UDPGA to a final concentration of 2 mM, or the addition of SAM to a final concentration of 400 μM, and the mixture was incubated at 37 °C for 30 min.[1]
For dehydroxylation, the 500 μL reaction mixture contains baicalein (final concentration, 300 μM), Tris-HCl (0.1 M, pH 7.4), MgCl2 (8 mM), trinatric isocitric acid (1.43 mg), and isocitric dehydrogenase (0.18 U). The reaction was initiated by the addition of NADP (0.9 mM) and NADPH (0.2 mM). The reaction was kept for 30 min at 37 °C (Xia et al., 2007).[1]
A total of 100 μL of reaction mixtures was taken out and the reaction was terminated by the addition of 100 μL acetonitrile/acetic acid (9:1, v/v, −20 °C), containing 74 μM IS. After centrifugation at 13000 × g for 10 min, the supernatant was collected and analyzed by HPLC-MS/MS.

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Protein concentration measurement[1]
Protein concentrations of rat S9 fractions were measured using a BCA protein assay kit, with bovine serum albumin (BSA) as the standard.

Intestinal pharmacokinetics of baicalein in mice[1]
Thirty-three KM mice were fasted for 16 h with free access to water and were then administrated with 60 mg/kg (222 μmol/kg) baicalein by oral gavage. Three mice were sacrificed at each time point, including 0, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, 24 h, and the gastrointestinal tissues were excised and divided into stomach, duodenum, jejunum, ileum, cecum, and colon. The extraction of baicalein metabolites in intestinal tissues was performed according previously published method with minor modification (Lu et al., 2012). After the addition of 1 mL of pre-chilled ethyl acetate and 20 μL of 0.58 M acetic acid solution, the tissues were cut into pieces, homogenized at 4 °C, and ultrasonicated for 5 min. After centrifugation at 12,000g for 10 min, the supernatant was evaporated and the pellet was dried at room temperature. The pellet was resuspended with 800 μL methanol containing 58 μM of acetic acid and 74 μM of internal standard (IS) luteolin. The samples were stored at −70 °C for the subsequent HPLC-MS analysis.

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Fecal pharmacokinetics of baicalein in mice[1]
Three KM mice had free access to both diet and water. After oral administration of 60 mg/kg baicalein, feces were checked and collected every 30 min in 0–12 h time frame; every hour in 12–24 h time frame; every 2 h in 24–60 h time frame, and were combined at 0, 2, 4, 6, 8, 12, 16, 20, 24, 30, 36, 48, and 60 h. The mice litter was frequently changed to maintain a completely dry condition. The extraction of baicalein metabolites in the feces was performed according to previously published method (Xia et al., 2012). Feces samples were weighted, homogenized with 9 volumes (v/w) of methanol containing 58 mM acetic acid and 74 μM IS at 4 °C and the mixture was ultrasonicated for 5 min. After centrifugation at 12000 × g for 10 min, the supernatant was collected and stored at −70 °C for the subsequent HPLC-MS analysis.

[1]. (2019). Intestinal metabolism of baicalein after oral administration in mice: Pharmacokinetics and mechanisms. Journal of Functional Foods, 54, 53-63.

Baicalein 6-O-glucoside has been reported in Cephalocereus senilis with data available.

C21H20O10

432.38

432.106

28279-72-3

5321896

Typically exists as solid at room temperature

1.642±0.06 g/cm3

0.049

6

10

4

31

677

5

C1=CC=C(C=C1)C2=CC(=O)C3=C(C(=C(C=C3O2)O)O[C@H]4[C@@H]([C@H]([C@@H]([C@@H](CO)O4)O)O)O)O

WTYYPLMAODUDGW-QOUKUZOOSA-N

InChI=1S/C21H20O10/c22-8-14-16(25)18(27)19(28)21(30-14)31-20-11(24)7-13-15(17(20)26)10(23)6-12(29-13)9-4-2-1-3-5-9/h1-7,14,16,18-19,21-22,24-28H,8H2/t14-,16-,18+,19-,21+/m1/s1

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

Baicalein 6-O-glucoside; 28279-72-3; Tetuin; BAicalein 6-O-; A-D-glucopyranoside;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)