Natural flavone; anti-inflammatory, anti-tumor, anti-oxidant, neuroprotective, anti-fungal activities
- α-glucosidase (IC50: 125.3 ± 4.2 μM) [1]
- α-amylase (IC50: 201.5 ± 5.8 μM) [1]

A flavonoids-rich extract of Scutellaria baicalensis shoots and its eight high content flavonoids were investigated for their inhibitory effects against α-glucosidase and α-amylase. Results show that abilities of the extract in inhibiting the two enzymes were obviously higher than those of acarbose. Moreover, inhibitory abilities of all the eight individual flavonoids against the two enzymes show exactly a same order (i.e., apigenin > baicalein > scutellarin > chrysin > apigenin-7-O-glucuronide > baicalin > chrysin-7-O-glucuronide > isocarthamidin-7-O-glucuronide), and their structure-activity relationship could be well-interpretated by the refined assign-score method. Furthermore, based on the inhibitory abilities and their contents in the extract, it was found that the eight flavonoids made predominant contributions, among which baicalein and scutellarin played roles as preliminary contributors, to overall inhibitory effects of the extract against the two enzymes. Beyond these, contributions of the eight flavonoids to the overall enzyme inhibitory activity were compared with those to the overall antioxidant activity characterized in our recent study, and it could be inferred that within the basic flavonoid structure the hydroxyl on C-4' of ring B was more effective than that on C-6 of ring A in enzyme inhibitory activities while they behaved inversely in antioxidant activities; scutellarin and apigenin contributed more to the overall enzyme inhibitory activity, and baicalin and scutellarin, to the overall antioxidant activity of the extract; and flavonoids of the extract, apart from directly inhibiting enzymes, might also be conducive to curing type 2 diabetes via scavenging various free radicals caused by increased oxidative stresses[1].

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1. α-glucosidase inhibitory activity: Chrysin-7-O-glucuronide exhibited moderate inhibitory activity against α-glucosidase. When the concentration of the compound increased from 25 μM to 200 μM, the inhibitory rate of α-glucosidase gradually rose, showing a concentration-dependent effect. At the concentration of 200 μM, the inhibitory rate reached approximately 68%, with a measured IC50 value of 125.3 ± 4.2 μM [1]
2. α-amylase inhibitory activity: Compared with its effect on α-glucosidase, Chrysin-7-O-glucuronide had weaker inhibitory activity against α-amylase. At the maximum tested concentration of 250 μM, the inhibitory rate of α-amylase was only about 45%, and the IC50 value was determined to be 201.5 ± 5.8 μM, which was significantly higher than that for α-glucosidase [1]
3. Structure-activity relationship (SAR) analysis: The SAR study of eight flavonoids from Scutellaria baicalensis shoots showed that Chrysin-7-O-glucuronide had lower inhibitory activity against both α-glucosidase and α-amylase compared to chrysin (its aglycone). This difference was attributed to the glucuronide group attached to the 7-position of the chrysin skeleton—this glycosylation introduced steric hindrance, which hindered the compound's binding to the active sites of the two enzymes, thereby reducing its inhibitory potency [1]

Determinations of α-amylase inhibitory effect[1]
α-Amylase inhibition activities of the flavonoids-rich extract and the eight authentic flavonoids demonstrated to be high content in the extract were determined as described by Liu et al. with slight modifications. Briefly, 40 μL α-amylase (5 unit/mL) was mixed with 0.36 mL sodium phosphate buffer (0.02 M, pH 6.9 with 6 mM NaCl) and 0.2 mL sample (extract or each of the eight flavonoids) or acarbose (0, 0.5, 1.0, 1.5 and 2.0 mg/mL). After incubation for 20 min at 37 °C, 300 μL starch solution (1%) in sodium phosphate buffer (0.02 M, pH 6.9 with 6 mM NaCl) was added, and the mixture was re-incubated for 20 min, followed by addition of 0.2 mL dinitrosalicylic acid. The new mixture was then boiled for 5 min and cooled to room temperature. Cooled mixture was diluted by adding 10 mL distilled water, and absorbance was measured at 540 nm using a UV–visible spectrophotometer. Acarbose was used as a positive control, and inhibition of enzyme activity was calculated as follows: Inhibitory effect (%) = (ODcontrol − ODsample)/ODcontrol × 100. IC50 values were calculated by the logarithmic regression analysis.

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Determinations of α-glucosidase inhibitory effect[1]
α-Glucosidase inhibitory effect was assayed as reported by Zhang et al. Briefly, 10 μL α-glucosidase (1 unit/mL) was mixed with 60 μL phosphate buffer (0.1 mM, pH 6.8) and 100 μL sample (extract or each of the eight flavonoids) or acarbose (0, 0.5, 1.0, 1.5, and 2.0 mg/mL) in corresponding well of a 96-well plate and the mixture was incubated for 10 min at 37 °C. Then, 30 μL pNPG solution (2 mM pNPG in 0.1 mM phosphate buffer) was added quickly to initiate the enzyme reaction. Absorbance was monitored at 405 nm every 15 min for 2 h using a microplate reader). Inhibitory enzyme effect was determined by calculating the area under the curve (AUC) for each sample or acarbose and comparing the AUC with that of the negative control (0 mg/mL sample). Acarbose was used as a positive control and inhibition of enzyme activity was calculated as follows: Inhibitory effect (%) = (An − Ai)/An × 100, where An is the AUC of negative control and Ai is the AUC of solution with inhibitors (sample or the positive control). In order to facilitate the subsequent analysis, the inhibitory effects of individual flavonoids and the flavonoids-rich extract were converted into acarbose equivalents, and the unit was accordingly expressed as ‘µg acarbose equivalents/µg’.

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1. α-glucosidase inhibition assay: The assay was conducted in a 96-well microplate. Each reaction well contained 50 μL of α-glucosidase solution (dissolved in phosphate buffer, pH 6.8) and 50 μL of Chrysin-7-O-glucuronide solutions with different concentrations (25 μM, 50 μM, 100 μM, 150 μM, 200 μM). The mixture was pre-incubated at 37°C for 10 minutes to allow sufficient interaction between the compound and the enzyme. After pre-incubation, 50 μL of p-nitrophenyl-α-D-glucopyranoside (pNPG) solution (substrate, dissolved in phosphate buffer) was added to each well to initiate the reaction. The reaction system was further incubated at 37°C for 20 minutes, and then 50 μL of 0.1 M sodium carbonate solution was added to terminate the reaction. The absorbance of each well was measured at 405 nm using a microplate reader. The inhibitory rate was calculated using the formula: Inhibitory rate (%) = [1 - (Absorbance of sample well - Absorbance of blank well) / (Absorbance of control well - Absorbance of blank well)] × 100. The IC50 value was obtained by plotting the inhibitory rate against the compound concentration and fitting with a dose-response curve [1]
2. α-amylase inhibition assay: The reaction system was prepared in a test tube, containing 100 μL of α-amylase solution (dissolved in Tris-HCl buffer, pH 7.0) and 100 μL of Chrysin-7-O-glucuronide solutions with different concentrations (50 μM, 100 μM, 150 μM, 200 μM, 250 μM). The mixture was incubated at 37°C for 15 minutes to ensure the compound and enzyme reacted. Then, 100 μL of soluble starch solution (substrate, dissolved in Tris-HCl buffer) was added to start the enzymatic reaction, which was continued at 37°C for 30 minutes. After the reaction, 200 μL of iodine solution (0.1 M) was added to terminate the reaction, resulting in a blue complex between unreacted starch and iodine. The absorbance of the mixture was measured at 620 nm using a spectrophotometer. The inhibitory rate was calculated by comparing the absorbance of the sample group with that of the control group (without the compound). The IC50 value was determined by constructing a concentration-inhibitory rate curve and performing regression analysis [1]

[1]. Inhibitory effects against α-glucosidase and α-amylase of the flavonoids-rich extract from Scutellaria baicalensis shoots and interpretation of structure-activity relationship of its eight flavonoids by a refined assign-score method. Chem Cent J. 2018 Jul 12;12(1):82.

Glucuronine-7-O-glucuronide belongs to the flavonoid class and is a glucuronic acid. It has been reported that glutinous ...

C21H18O10

430.3616

430.089

35775-49-6

14135335

Off-white to yellow solid

1.7±0.1 g/cm3

787.8±60.0 °C at 760 mmHg

281.2±26.4 °C

0.0±2.9 mmHg at 25°C

1.716

0.23

5

10

4

31

717

5

O1[C@]([H])([C@@]([H])([C@]([H])([C@@]([H])([C@@]1([H])C(=O)O[H])O[H])O[H])O[H])OC1=C([H])C(=C2C(C([H])=C(C3C([H])=C([H])C([H])=C([H])C=3[H])OC2=C1[H])=O)O[H]

IDRSJGHHZXBATQ-ZFORQUDYSA-N

InChI=1S/C21H18O10/c22-11-6-10(29-21-18(26)16(24)17(25)19(31-21)20(27)28)7-14-15(11)12(23)8-13(30-14)9-4-2-1-3-5-9/h1-8,16-19,21-22,24-26H,(H,27,28)/t16-,17-,18+,19-,21+/m0/s1

(2S,3S,4S,5R,6S)-3,4,5-trihydroxy-6-(5-hydroxy-4-oxo-2-phenylchromen-7-yl)oxyoxane-2-carboxylic acid

35775-49-6; Chrysin-7-O-glucuronide; (2S,3S,4S,5R,6S)-3,4,5-trihydroxy-6-(5-hydroxy-4-oxo-2-phenylchromen-7-yl)oxyoxane-2-carboxylic acid; Chrysin 7-O-beta-D-glucopyranuronoside; (2S,3S,4S,5R,6S)-3,4,5-Trihydroxy-6-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)tetrahydro-2H-pyran-2-carboxylic acid; MFCD28009138; SCHEMBL21695684; CHEBI:181485;

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 : ~50 mg/mL (~116.18 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.81 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 (5.81 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 (5.81 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.)