Endothelium-derived nitric oxide (NO) pathway (effect inhibited by L-NAME, an NO synthase inhibitor) [2]

In rat thoracic aortic rings with functional endothelium precontracted with norepinephrine, delphinidin chloride caused endothelium-dependent vasorelaxation. The maximal relaxation (Emax) was 83.86 ± 4.87% (or 80.27 ± 5.64% in another dataset), and the molar concentration producing half-maximal relaxation (EC50) was 26.30 ± 0.41 μmol/L. This vasorelaxant effect was completely inhibited by pre-incubation with L-NAME (300 μmol/L), indicating the involvement of nitric oxide. [2]

Cell viability was assessed by MTT assay. HCT116 cells were seeded at 10,000 cells/well in 96-well plates and treated with various concentrations of Delphinidin for 48 h. MTT solution (100 μL, 5 mg/mL) was added for 4 h, then formazan crystals were dissolved in 100 μL DMSO, and absorbance was recorded at 570 nm. [3]
ROS measurement: Cells were seeded in 6-well plates, treated with Delphinidin for 48 h, then stained with DCFH-DA (1 mg/mL) for 30 min in the dark. After washing with PBS, fluorescence intensity was recorded at excitation 485±10 nm and emission 530±12.5 nm, and images were captured at 40× magnification. [3]
Mitochondrial membrane potential (MMP) assessment: Cells were treated as above, then stained with Rhodamine 123 (Rh-123, 1 mg/mL) for 30 min in the dark. Fluorescence intensity was recorded at excitation 488±10 nm and emission 530±12.5 nm, and images were captured at 40×. [3]
Lipid peroxidation was measured using the colorimetric method of Niehaus and Samuelsson. [3]
Antioxidant levels: Superoxide dismutase (SOD) was evaluated by Kakkar et al. method; catalase (CAT) by Sinha method; glutathione (GSH) by Rotruck et al. method. [3]
DNA damage (comet assay): After treatment, cells were mixed with low melting agarose and coated on slides with normal melting agarose. Slides were kept in cold lysis solution (pH 10) for 1 h at 4°C, then placed in alkaline electrophoresis buffer and electrophoresed for 25 min at 25 V. After neutralization with 0.4 M Tris (pH 7.5) for 5 min, slides were stained with ethidium bromide and images captured at 40×, analyzed by CASP software. [3]
Apoptosis morphological changes (AO/EtBr staining): Treated cells were incubated with AO/EtBr (1 mg/mL, 5 μL) for 30 min in the dark, washed with PBS, and images taken at 40×. [3]
Acridine orange staining for DNA damage: Treated cells were incubated with acridine orange (1 mg/mL, 5 μL) for 30 min in the dark, washed with PBS, and images taken at 40×. [3]
Hoechst staining for nuclear condensation: Treated cells were incubated with Hoechst stain (1 mg/mL, 1 μL) for 30 min in the dark, washed with PBS, and images taken at 40×. [3]
Western blot analysis: Cells with or without Delphinidin treatment were lysed with RIPA buffer containing protease inhibitor cocktail. Proteins were fractionated on 10% SDS-PAGE, transferred to nitrocellulose membranes, probed with primary antibodies against STAT-3, phosphor-STAT-3 (Tyr705), phospho-ERK1/2 (Thr202/Tyr204), JAK2, phosphor-JAK2 (Tyr1007/1008), phospho-p38 (Thr180/Tyr182), Bcl-2, Bad, cytochrome C, Bax, Bcl-XL, caspase-3, caspase-8, caspase-9, and beta-actin, then with HRP-conjugated secondary antibodies. Blots were developed by chemiluminescence. [3]

Male Wistar rats (12–14 weeks old) were killed by cervical dislocation and exsanguinated by carotid artery transection. The thoracic aorta was removed, cleaned of adhering fat and connective tissue, and cut into rings (2–3 mm length). Rings with functional endothelium were mounted in organ baths filled with physiological salt solution (composition in mmol/L: NaCl 119, KCl 4.7, CaCl2 1.25, MgSO4 1.17, KH2PO4 1.18, NaHCO3 25, glucose 11), maintained at 37°C and continuously bubbled with 95% O2–5% CO2. Resting tension was adjusted to 2 g. After an equilibration period of 90 min, vessels were precontracted with norepinephrine (0.3 μmol/L) to about 80% of maximal response. Then, cumulative concentrations of delphinidin chloride (10−5–3×10−1 g/L, corresponding to 3.0×10−8–0.9×10−3 mol/L as malvidin equivalent) were added. To study the involvement of endothelial NO, some arteries were exposed to the NO synthase inhibitor L-NAME (300 μmol/L) for 15 min prior to contraction with norepinephrine. [2]

---

Male Wistar rats (12–14 weeks old) were killed by cervical dislocation and exsanguinated by carotid artery transection. The thoracic aorta was removed, cleaned of adhering fat and connective tissue, and cut into rings (2–3 mm length). Rings with functional endothelium were mounted in organ baths filled with physiological salt solution (composition in mmol/L: NaCl 119, KCl 4.7, CaCl2 1.25, MgSO4 1.17, KH2PO4 1.18, NaHCO3 25, glucose 11), maintained at 37°C and continuously bubbled with 95% O2–5% CO2. Resting tension was adjusted to 2 g. After an equilibration period of 90 min, vessels were precontracted with norepinephrine (0.3 μmol/L) to about 80% of maximal response. Then, cumulative concentrations of delphinidin chloride (10−5–3×10−1 g/L, corresponding to 3.0×10−8–0.9×10−3 mol/L as malvidin equivalent) were added. To study the involvement of endothelial NO, some arteries were exposed to the NO synthase inhibitor L-NAME (300 μmol/L) for 15 min prior to contraction with norepinephrine. [2]

Toxicity Data
LC50 (Rat) = 2,062 ppm/4h

---

[1]. Determination of anthocyanidins in berries and red wine by high-performance liquid chromatography. J Agric Food Chem. 2001 Sep;49(9):4183-7.

[2]. Natural dietary polyphenolic compounds cause endothelium-dependent vasorelaxation in rat thoracic aorta. J Nutr. 1998 Dec;128(12):2324-33.

[3]. Delphinidin modulates JAK/STAT3 and MAPKinase signaling to induce apoptosis in HCT116 cells. Environ Toxicol. 2021 May 6.

[4]. The anthocyanidins cyanidin and delphinidin are potent inhibitors of the epidermal growth-factor receptor. J Agric Food Chem. 2001 Feb;49(2):958-62.

Delphinidin chloride is an anthocyanin chloride, and its cationic counterpart is delphinidin. It contains delphinidin.
See also: Paraffin (note moved to).

---

Delphinidin chloride is an anthocyanin aglycone. Among the tested defined polyphenols (including phenolic acid derivatives, hydroxycinnamic acids, flavanols, and other anthocyanins such as malvidin and cyanidin), only delphinidin chloride elicited endothelium-dependent vasorelaxation comparable to that of the original red wine polyphenolic compounds (RWPC). It is suggested that anthocyanins like delphinidin may contribute to the cardioprotective effects of fruits, vegetables, and red wine by increasing endothelial nitric oxide production. [2]

C15H11CLO7

338.6966

338.019

528-53-0

528-53-0;528-53-0 (Cl);

68245

Brown to black solid powder

1.3946 (rough estimate)

454.94°C (rough estimate)

>350ºC

1.4429 (estimate)

6

7

1

23

380

0

[Cl-].[O+]1C2=C([H])C(=C([H])C(=C2C([H])=C(C=1C1C([H])=C(C(=C(C=1[H])O[H])O[H])O[H])O[H])O[H])O[H]

FFNDMZIBVDSQFI-UHFFFAOYSA-N

InChI=1S/C15H10O7.ClH/c16-7-3-9(17)8-5-12(20)15(22-13(8)4-7)6-1-10(18)14(21)11(19)2-6;/h1-5H,(H5-,16,17,18,19,20,21);1H

2-(3,4,5-trihydroxyphenyl)chromenylium-3,5,7-triol;chloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.  (2). This product is not stable in solution, please use freshly prepared working solution for optimal results.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~62.5 mg/mL (~184.53 mM)

Solubility in Formulation 1: 2.5 mg/mL (7.38 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (6.14 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 20.8 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.

---

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