Natural product from Spica Prunellae

The antioxidant activity of P. vulgaris spicas showed a similar fluctuation with growth stage as was seen in the phenolic and flavonoid contents since the DPPH● and ABTS•+ scavenging activities were positively correlated with the contents of bioactive compounds in the spica except in case of the Salviaflaside [1].

Determination of the Caffeic Acid, Salviaflaside, Rosmarinic Acid and Hyperoside Contents [1]
The powder of dried spica (2.0 g) was mixed with 80% methanol (15 mL) in an ultrasonic bath for 35 min at room temperature, and then the extracted solution was centrifuged at 12,000 rpm for 15 min. The supernatant was passed through a 0.45-μm organic membrane filter before HPLC analysis. The extract (10 μL) was analysed on a Dionex UltiMate 3000 HPLC System equipped with a Uranus C18 (250 mm × 4.6 mm) and a diode array detector (DAD-3000). The mobile phase consisted of methanol (solvent A) and 0.2% NaH2PO4 solution (solvent B), the flow rate was 0.8 mL min−1 and the following multilinear gradient was used: 20–40% A (0–20 min), 40–70% A (20–35 min), 70–90% A (35–45 min), and 90–20% A (45–60 min). The detection wavelengths were 325 nm and 360 nm (325 nm detection wavelength for caffeic acid, Salviaflaside and rosmarinic acid and 360 nm detection wavelength for hyperoside). The column oven temperature was 30 °C, and the total run time was 60 min. Each peak was identified based on its retention time and comparison to chromatographs of authentic standards. The contents of the bioactive compounds were calculated according to calibration curves of the standards (Table 3), and the results are expressed as mg per 100 mg dry weight of spicas (%). HPLC chromatograms of the mixed standard stock solution and an extract of P. vulgaris are shown in Figure 3.

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DPPH Free Radical Scavenging Assay [1]
The antioxidant scavenging activity was studied using the DPPH● spectrophotometric method described in a previous study with some modifications. The dried powder of spica (1.0 g) was extracted with 70% ethanol (30 mL) using ultrasound extraction for 30 min at 70 °C. An aliquot of the sample solution (1.0 mL) was added to 4 mL of a methanolic solution of DPPH● (0.004%, w/v, 50 μg mL−1). Meanwhile, a 1.0 mL aliquot of the sample solution was added to 4 mL of methanol as the blank, and a 1.0 mL aliquot of methanol was added to 4 mL of methanolic DPPH● as the control. After incubation at 25 °C for 30 min, the absorbance was measured at 517 nm using a 752 UV-visible spectrophotometer. All measurements were performed in triplicate and were taken while protected from light. The DPPH● scavenging effect was calculated as follows:
DPPH● scavenging effect (%) = [Acontrol − (Asample − Ablank)/Acontrol] × 100%
where Acontrol is the absorbance of 4 mL of DPPH● radical + 1 mL of methanol, Asample is the absorbance of 4 mL of DPPH• radical + 1 mL of sample, and Ablank is the absorbance of 4 mL of methanol + 1 mL of sample.

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Trolox Equivalent Antioxidant Capacity Assay (TEAC Assay) [1]
The radical scavenging capacity for the ABTS•+ radical cation was determined following the procedure previously reported with some modifications. The radical was generated by reacting a 7 mmol L−1 solution of ABTS•+ with 2.45 mmol L−1 potassium persulfate. The final working solution of ABTS•+ was obtained after allowing this mixture to react for 12 to 16 h at ambient temperature (23 °C) in darkness. The ABTS•+ reagent was diluted with phosphate buffered saline (pH 7.4) to reach an absorbance of 0.700 ± 0.005 at 734 nm. For the spectrophotometric measurements of the samples, 3.9 mL of dilute ABTS•+ solution and 0.1 mL of sample were mixed. A calibration curve was prepared using Trolox at concentrations ranging from 80 to 1600 μmol L−1 in methanol. Dilute ABTS•+ solution (3.9 mL) mixed with 0.1 mL of water was used as the control. The absorbance was measured at 734 nm after 6 min using a 752 UV-visible spectrophotometer. The total antioxidant capacities of the samples are expressed as equivalents of Trolox (TEAC) per 1 g of dry matter of the sample (μmol g−1). All measurements were performed in triplicate, and the ABTS•+ scavenging effect was calculated as follows:
ABTS•+ scavenging effect (%) = [(1 − Asample)/Acontrol] × 100% where Asample is the absorbance of 3.9 mL of diluted ABTS•+ + 0.1 mL of sample and A control is the absorbance of 3.9 mL of diluted ABTS•+ + 0.1 mL of water.
Trolox was used to prepare a standard calibration curve, and the results are expressed as TEAC. The concentration of antioxidants giving the same percentage inhibition of ABTS•+ as that achieved with 1 mM Trolox was regarded as the TEAC.

[1]. Effects of UV-B Radiation on the Content of Bioactive Components and the Antioxidant Activity of Prunella vulgaris L. Spica during Development. Molecules. 2018 Apr 24;23(5).

Salviaflaside is a glycoside.
Salviaflaside has been reported in Perilla frutescens, Salvia deserti, and other organisms with data available.

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The effects of UV-B radiation on the content of bioactive components and the antioxidant activity of Prunella vulgaris L. spica during development were studied. The experimental design involved two levels of UV-B radiation intensity (0 and 120 μW cm-2 nm-1). The results showed that the contents of total flavonoids, rosmarinic acid, caffeic acid and hyperoside, as well as the antioxidant capacities (DPPH● and ABTS•+ scavenging activities), in the spicas significantly decreased during spica development. The content of Salviaflaside in the spicas significantly increased during development. The highest contents of total flavonoids, rosmarinic acid, and caffeic acid and the highest antioxidant activities were found in spicas in the full-flowering stage, while the highest content of hyperoside was found in spicas in the bud stage. In addition, the highest content of Salviaflaside was found in spicas in the mature-fruiting stage. UV-B radiation significantly promoted the synthesis of secondary metabolites, increased the contents of the main bioactive components in the three developmental stages of isolated dried spicas, and significantly increased the DPPH● and ABTS•+ scavenging activities of P. vulgaris spicas in the mature-fruiting stage. Moreover, the total flavonoids content was positively correlated with the DPPH● and ABTS•+ scavenging activities, and the correlation with the DPPH● scavenging activity was very strong. This result shows that the highest contents of the main bioactive components in the spicas were not all found in the same developmental stages of P. vulgaris. Our research revealed that the best stage for harvesting P. vulgaris spica was between the bud stage and the full-flowering stage since harvesting at this point provides a higher content of bioactive components and a higher antioxidant capacity, which is relevant for medicinal applications. [1]

C24H26O13

522.4554

522.137

178895-25-5

6438919

White to off-white solid

1.625g/cm3

873.2ºC at 760 mmHg

296.6ºC

1.35E-32mmHg at 25°C

1.706

0.6

8

13

10

37

791

6

O1[C@]([H])([C@@]([H])([C@]([H])([C@@]([H])([C@@]1([H])C([H])([H])O[H])O[H])O[H])O[H])OC1=C(C([H])=C([H])C(/C(/[H])=C(\[H])/C(=O)O[C@@]([H])(C(=O)O[H])C([H])([H])C2C([H])=C([H])C(=C(C=2[H])O[H])O[H])=C1[H])O[H]

DSMWJDJWYGMEBO-PRFRQLEPSA-N

InChI=1S/C24H26O13/c25-10-18-20(30)21(31)22(32)24(37-18)36-16-8-11(1-5-14(16)27)3-6-19(29)35-17(23(33)34)9-12-2-4-13(26)15(28)7-12/h1-8,17-18,20-22,24-28,30-32H,9-10H2,(H,33,34)/b6-3+/t17-,18-,20-,21+,22-,24-/m1/s1

(2R)-3-(3,4-dihydroxyphenyl)-2-[(E)-3-[4-hydroxy-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyphenyl]prop-2-enoyl]oxypropanoic acid

Salviaflaside; 178895-25-5; (2R)-3-(3,4-dihydroxyphenyl)-2-[(E)-3-[4-hydroxy-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyphenyl]prop-2-enoyl]oxypropanoic acid; (R-(E))-alpha-((3-(3-(beta-D-glucopyranosyloxy)-4-hydroxyphenyl)-1-oxo-2-propenyl)oxy)-3,4-dihydroxybenzenepropanoic acid; Benzenepropanoic acid, alpha-((3-(3-(beta-D-glucopyranosyloxy)-4-hydroxyphenyl)-1-oxo-2-propenyl)oxy)-3,4-dihydroxy-, (R-(E))-; (2R)-3-(3,4-dihydroxyphenyl)-2-((E)-3-(4-hydroxy-3-((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl)oxyphenyl)prop-2-enoyl)oxypropanoic acid; Salviaflaside (Standard);

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.)