Natural flavonoid

The growing demand for the medicinal plant, Hypericum perforatum, is not being met by available supply in many countries. The conditions for obtaining quality crude drug should be thoroughly studied to ensure adequate supply. The current study describes the variation of major bioactive compounds in different accessions of H. perforatum (Hypericaceae) in two floral development stages. Two extraction procedures were evaluated for the quantification of the major bioactive compounds in H. perforatum. The Soxhlet extraction technique was demonstrated to be more effective with higher extraction yields. The contents of rutin, hyperoside, quercitrin, quercetin, and 3,8″-biapigenin were determined by high performance liquid chromatography (HPLC). This procedure was used to establish the variation in the contents of flavonoids in the field-growing H. perforatum and to evaluate factors predetermining that variation with some practical implications for utilization. A significant difference in flavonoid contents between two floral development stages was detected. The results of the study showed that rutin prevailed during the budding stage, whereas quercitrin, quercetin, and 3,8″-biapigenin were dominant during the full-flowering stage. The contents of hyperoside were similar in both developmental stages of the plants. The highest amounts of the most bioactive compounds were observed in the full-flowering stage, which could therefore be considered the best stage for the harvesting of the raw material of H. perforatum, known as Hyperici herba in pharmaceutics. The study revealed evident intraspecific variations in the flavonoid contents of H. perforatum. Several chemotypes were recognized by qualitative and quantitative differences in flavonoids. Qualitative analysis of the flavonoid contents confirmed the presence of the chemotype of H. perforatum containing no rutin, though the chemotype with higher levels of both rutin and hyperoside was more frequent. The accessions of H. perforatum also showed remarkable differences in chemical composition depending on the origin of plants. Therefore, employing the correct developmental stage for the harvesting of the selected accessions of H. perforatum is highly recommended for the quality production of the drug Hyperici herba. [1]

Standard solution [1]
Standard 3,8″-biapigenin was isolated and purified by partition and by column chromatography. The following standard compounds were used for comparative identification and quantitative determination: rutin, hyperoside, quercitrin, and quercetin. A reference standard of flavonoids was dissolved in HPLC-grade methanol. The calibration standard working solutions were freshly prepared by diluting the stock solution with methanol in appropriate quantities. The standard curves were obtained by plotting the peak areas of standard concentrations of rutin and hyperoside (0.5–500 μg ml−1), quercitrin and quercetin (0.5–100 μg ml−1) and 3,8″-biapigenin (0.5–125 μg ml−1). The concentration of flavonoids was expressed as mg/g dry matter (DM). Values represent the mean of analysis of three independently extracted samples.

[1]. Variation in concentrations of major bioactive compounds in Hypericum perforatum L. from Lithuania. Industrial Crops & Products, 2012, 35(1):302-308.

4',4''',5,5'',7,7''-Hexahydroxy-3,8''-biflavone is a flavonoid oligomer.
3,8'-biapigenin has been reported in Garcinia livingstonei, Hypericum perforatum, and other organisms with data available.

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The accurate quantification of major bioactive compounds in H. perforatum accessions is dependent on the selection of appropriate extraction technique and conditions. Our results demonstrated that greater quantities of flavonoids could be obtained by Soxhlet extraction than by maceration. Our results indicate that plant material at different floral developmental stages contain different contents of bioactive compounds. The highest accumulation of rutin was observed in floral budding stage, while higher quantities of quercitrin, quercetin and 3,8″-biapigenin were found in full-flowering stage. The content of hyperoside did not differ significantly between the stages. The relatively high rutin and hyperoside contents in some H. perforatum accessions established in this study should act as encouragement for the introduction of new cultivars of the species. The careful selection of populations and appropriate harvesting time provides opportunities for improved quality production of the crude drug Hyperici herba.

C30H18O10

538.46

538.09

C, 66.92; H, 3.37; O, 29.71

101140-06-1

10414856

Light yellow to yellow solid powder

1.7±0.1 g/cm3

911.7±65.0 °C at 760 mmHg

259 - 261 °C

308.8±27.8 °C

0.0±0.3 mmHg at 25°C

1.802

5.55

6

10

3

40

1050

0

C1=CC(=CC=C1C2=CC(=O)C3=C(O2)C(=C(C=C3O)O)C4=C(OC5=CC(=CC(=C5C4=O)O)O)C6=CC=C(C=C6)O)O

IQAMTZLKUHMPPE-UHFFFAOYSA-N

InChI=1S/C30H18O10/c31-15-5-1-13(2-6-15)22-12-21(37)24-19(35)11-20(36)26(30(24)39-22)27-28(38)25-18(34)9-17(33)10-23(25)40-29(27)14-3-7-16(32)8-4-14/h1-12,31-36H

3-[5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxochromen-8-yl]-5,7-dihydroxy-2-(4-hydroxyphenyl)chromen-4-one

3,8'-Biapigenin; 101140-06-1; 3,8''-biapigenin; biapigenin; 13,Ii8-Biapigenin; [3,8'-Bi-4H-1-benzopyran]-4,4'-dione, 5,5',7,7'-tetrahydroxy-2,2'-bis(4-hydroxyphenyl)-; CHEMBL515252; 3-[5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxochromen-8-yl]-5,7-dihydroxy-2-(4-hydroxyphenyl)chromen-4-one;

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.)