Natural flavonoid; aldose reductase

The purpose of this study is to evaluate the active properties of Thuja orientalis leaves for the treatment of diabetic complications. The ethyl acetate fraction showed a significantly higher inhibition both of recombinant human aldose reductase (rhALR2) inhibitory activity and advanced glycation endproducts (AGEs). The detected antioxidants (compounds 1–4) by online-HPLC-ABTS+ method and other three compounds were isolated using preparative RP-HPLC and Sephadex LH-20 column chromatography. Among the seven compounds, compound 4 (quercitrin) which was isolated from ethyl acetate fraction, was found to show inhibition for both forms of rhALR2 and AGEs. This compound also inhibited oxidative stress which was measured by Photochem® apparatus. In conclusion, the ethyl acetate fraction from T. orientalis demonstrated antioxidant activity as well as inhibitory effects on rhALR2 and AGEs. Quercitrin was shown to be the active compound and hence could be offered as an active material of standardization for the development of natural products for food or medicines [1].

Measurement of rhALR2 activity. rhALR2 activities were assayed spectrophotometrically by measuring the decrease in absorption of NADPH at 340 nm over a 5 min period with DL-glyceraldehyde as a substrate [Sato and Kador, 1990]. Each 1.0 mL cuvette contained equal units of enzyme, 0.10 M sodium phosphate buffer (pH 6.2), 0.3 mM NADPH with or without 10 mM substrate and inhibitor. The concentration of inhibitors giving 50% inhibition of enzyme activity (IC50) was calculated from the least-squares regression line of the logarithmic concentrations plotted against the residual activity.[1]

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Determination of inhibition-type of rhALR2 by the compounds. Reaction mixtures consisted of 0.1M potassium phosphate (pH 7.0), 0.16 mM NADPH, 2 μM of rhALR2 with varied concentrations of substrate DLglyceraldehyde and ALR2 inhibitors, epalrestat or quercitrin in a total volume of 200 μL. Concentrations were ranged from 0.02 mM to 0.2 mM for DL-glyceraldehyde, from 0.5 μM to 1.5 μM for epalrestat, and from 1.0 μM to 2.0 μM for quercitrin. rhALR2 activity was assayed spectrophotometrically by measuring the decrease in absorption of NADPH at 340 nm after substrate addition using BioTek Power Wave XS spectrophotometer.[1]

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ects on AGEs formation. The AGEs formation was assessed by characteristic fluorescence reported by Morimitsu et al. [1995] with slight modifications. Briefly, the reaction mixture of 100 mg D-glucose, 10 mg BSA in 1 mL sodium phosphate buffer (100 mM, pH 7.4) was incubated at 60oC for 2 days with or without the test compound. The reaction solution (0.2 mL) was diluted with water (2 mL), and the intensity of fluorescence was measured using a fluorophotometer at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The reaction mixture without Dglucose was used as a blank solution.[1]

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Antioxidative capacity determination. For the determination of the integral antioxidative capacity of lipid soluble substances in T. orientalis, the method of photochemiluminescence was used. ACL, where the luminol plays a double role as photosensitizer and also as oxygen radical detection reagent was used according to the manufacturer’s instruction [Jung et al., 2007]. Results were calculated in Trolox equivalents[1].

[1]. Eun Ha Lee, Flavonoids from the leaves of Thuja orientalis inhibit the aldose reductase and the formation of advanced glycation endproducts. Journal of the Korean Society for Applied Biological Chemistry volume 52, pages448–455 (2009).

Inosin 7-O-β-xyloside has been reported in Libocedrus bidwillii, Juniperus communis, and Juniperus communis var. depressa, with relevant data available. To quantitatively determine the antioxidant activity of ethyl acetate extracts and quercetin, a Photochem® instrument was used. This instrument utilizes a photosensitizer to generate free radicals. The generated free radicals are detected by their reaction with the chemiluminescent substance luminol. Therefore, luminol can be used as both a photosensitizer and a detection reagent [Apati et al., 2003]. If an antioxidant is added to this measurement system, the intensity of the photochemiluminescence signal decreases with concentration. Thus, the total antioxidant capacity of the studied sample can be quantified. Antioxidant capacity is expressed in Trolox equivalent units for lipid-soluble substances. The antioxidant capacity of the ethyl acetate extract of this plant was 1.66, 0.49 and 0.46 nmol at concentrations of 1000.0, 100.0 and 10.0 ng/mL, respectively. The antioxidant capacity of quercetin isolated from this plant was 4.53, 1.91 and 0.46 nmol at concentrations of 625.0, 125.0 and 62.5 ng/mL, respectively (Table 3). Therefore, the overall results indicate that the ethyl acetate extract of Smilax glabra has antioxidant activity as well as ALR2 and AGEs inhibition. In particular, quercetin isolated from the ethyl acetate extract of this plant may be active. Smilax glabra should be further studied in order to develop it into a novel natural product source for the treatment of diabetic complications. In addition, quercetin can be used as a standardized active substance for the development of food or pharmaceutical natural products. [1]

C20H18O11

434.35

434.084

126771-28-6

21589942

Typically exists as solid at room temperature

0.2

7

11

3

31

699

4

C1[C@H]([C@@H]([C@H]([C@@H](O1)OC2=C(C3=C(C(=C2)O)C(=O)C=C(O3)C4=CC(=C(C=C4)O)O)O)O)O)O

JZTWSAIHBOFVRO-MINVPOHDSA-N

InChI=1S/C20H18O11/c21-8-2-1-7(3-9(8)22)13-4-10(23)15-11(24)5-14(17(27)19(15)30-13)31-20-18(28)16(26)12(25)6-29-20/h1-5,12,16,18,20-22,24-28H,6H2/t12-,16+,18-,20+/m1/s1

2-(3,4-dihydroxyphenyl)-5,8-dihydroxy-7-[(2S,3R,4S,5R)-3,4,5-trihydroxyoxan-2-yl]oxychromen-4-one

HYPOLETIN-7-O-BETA-D-XYLOPYRANOSIDE; 126771-28-6; Hypoletin-7-O-; A-D-xylopyranoside;

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.)