Delphinidin

Extraction of anthocyanidins was performed as previously described with a slight modification [28 (link)]. The fresh petals were first freeze-dried and ground into fine powder. Then, the resulted flour was macerated with methanol:water:formic acid:TFA (70:27:2:1, v/v) and left to rest in the dark at 4 °C for 24 h. The extracted fluid was filtered with 0.22 μm membrane filter and used in subsequent experiments. Hydrolysis analysis of anthocyanins was performed according to Huang [29 (link)] and Morit [30 (link)]. An aliquot of 300 μL sample concentrations mentioned in HPLC analyses was transferred to a fresh tube, acid-hydrolyzed by adding 300 µL of 6 M HCL, incubated at 90 °C for 1 h. Hydrolyzation solution was immediately cooled to room temperature, and then was filtered prior to injection for analysis.
HPLC analysis used a Waters 600 series high-performance liquid chromatograph (Waters, Milford, MA, USA), a Waters 2487 UV detector (Waters, Milford, MA, USA), an Azbil ADC15 column oven (Azbil, Sanbu, Japan), a Rheodyne 7725i manual injector (Rheodyne of IDEX, Chicago, IL, USA), and a Waters Empower Build 1154-C software (Waters, Milford, MA, USA). An aliquot of 300 µL sample concentrations mentioned in HPLC analyses was transferred to a fresh tube, acid-hydrolyzed by adding 300 μL of 6 M HCL, incubated at 90 °C for 1 h. Hydrolyzation solution was immediately cooled to room temperature, and then was filtered through a 0.22 μm filter membrane prior to injection for analysis. The chromatographic column was a C18 TSK gel ODS-80Ts QA (250 mm × 4.6 mm i.d., 5 μm) (Tosoh, Yokkaichi, Japan), with a flow rate of 0.8 mL/min and injection amount of 10 μL. Mobile phase A employed a 0.1% formic acid solution and phase B an 80% acetonitrile solution. The elution programme was as follows: 0 min, 88% A, 12% B; 15 min, 75% A, 25% B; 32 min, 62% A, 38% B; 40 min, 62% A, 38% B; 45 min, 88% A, 12% B; 50 min, 88% A, 12% B. The detection wavelength was 530 nm. Results of HPLC were verified by at least three independent experiments. Cy, Dp, delphinidin-3-O-glucoside (Dp3G), malvidin-3-O-glucoside (Mv3G), Pg, petunidin-3-O-glucoside (Pt3G) were purchased from ChromaDex (Santa Ana, CA, USA). The concentration of anthocyanidins was quantified by external reference methods using Dp-chloride as standards.

RAW264.7 cells, a mouse macrophage cell line, were used as osteoclast precursor cells and maintained in α modified essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO₂. For osteoclast induction, cells were plated in a 96-well plate at a density of 4×10³ cells/well and stimulated with 100 ng/ml RANKL for 4 days. For the inhibition study, cells were pre-incubated in α-MEM supplemented with vehicle or with various concentrations of anthocyanin-rich extracts and anthocyanidins, 1 h before the addition of RANKL. To confirm multinucleated osteoclast formation, the cultured cells were fixed in 10% formalin for 3 minutes, and then stained with an osteoclast marker enzyme, tartrate-resistant acid phosphatase (TRAP). Effects of anthocyanins and anthocyanidins on osteoclast formation were evaluated by morphological observations and the intensity of TRAP staining was measured at 520 nm using a spectrophotometer (SpectraMax M5; Molecular Devices, Sunnyvale, CA, USA).
Osteoblasts were isolated from newborn calvariae of C57BL/6J mice, as described previously with slight modifications [19] (link). Briefly, calvariae were minced and sequentially digested with collagenase solution at 37°C. Cells retrieved from the osteogenic cell fractions were separately cultured in α-MEM supplemented with 10% FBS and antibiotics. After 24 h, cells were pooled and grown in multi-well plates in the same medium containing 50 µg/ml of ascorbic acid (AA), 10 µM dexamethasone (Dex) and 10 mM β-glycerophosphate (β-GP) with or without anthocyanin-rich extracts. After two weeks culture, cells were stained with von Kossa’ s staining to determine the matrix mineralization, as described previously [19] (link).

Anthocyanin mixtures from 30 DAA fruits of four pepper varieties (HN191, HN192, HN005, and EJT, respectively) were extracted and measured according to the agricultural standard of China (NY/T 2640-2014). The anthocyanin standard included delphinidin, cyanidin, petunidin, pelargonidin, peonidin, and malvidin, which were dissolved in methanol with 10% hydrochloric acid.

Epigallocatechin gallate (E4143), daidzein (D7802), delphinidin chloride (43725), catechin (43412), apigenin (10798), kaempferol (60010), genistein (G6649), quercetin (Q4951), and hesperetin (W431300) were obtained from Sigma Aldrich (St. Louis, MO). Naringenin (S2394) and nobiletin (S2333) were obtained from Selleck Chemicals (Houston, TX), and cyanidin chloride (sc-202559) was from BioMed (Heidelberg, Germany). Most flavonoids had a purity above 98%. However, epigallocatechin gallate, delphinidin, apigenin, hesperetin, and quercetin had a purity above 95%. The flavonoids were dissolved in dimethyl sulfoxide (DMSO; Sigma Aldrich), contained under argon gas and added to cells in a low light environment. Actinomycin D (A1410), cycloheximide, and dithiothreitol (D0632) were obtained from Sigma Aldrich. Antibody against LDLR (3839-100) was purchased from BioVision (Milpitas, CA), and antibody against β-actin (AB8227) was from Abcam (Cambridge, UK). Anti-LDLR IgG-C7 (#61087) used in flow cytometry was purchased from Progen Biotechnik GmbH (Heidelberg, Germany), while antibody against Poly (ADP-ribose) polymerase (PARP) was obtained from Cell signaling (Danvers, MA), and staurosporine was from Sigma-Aldrich (S4400). 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay kit was purchased from Abcam (ab21091), and Triton X-100 was from Sigma-Aldrich.