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CAS:153-18-4
Appearance:Light yellow to yellow powder
Storage:2-8℃
Purity:HPLC≥98%
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LC-ESI-MS/MS
The chromatographic conditions were as follows: BEH C18 chromatographic column (150?mm?×?2.1?mm, 1.7 μm); mobile phase: A phase water, B phase acetonitrile, 0.1% (vol/vol) formic acid and 0.2 mM ammonium acetate were added t both phases. Step-stripping sequence: 0–1.0 min, 10% B; 0–9.0?min, 10% B–90% B; 9.0–11.0?min, 90% B–100% B; 11.0–11.1?min, 100% B–10% B; 11.1–13.0 min, 10% B. Column temperature: 30℃; injection volume: 2 μl; flow rate: 0.25?ml/min. Mass spectrum conditions: retention time 4.94?min?1; ion pair l: quantitative ion 433.1/271.1, collision energy ?22?V; ion pair 2: quantitative ion 433.1/150.9, collision energy ?42?V.
References：
Sun M, Li L, Wang C, Wang L, Lu D, Shen D, Wang J, Jiang C, Cheng L, Pan X, Yang A, Wang Y, Zhu X, Li B, Li Y, Zhang F. Naringenin confers defence against Phytophthora nicotianae through antimicrobial activity and induction of pathogen resistance in tobacco. Mol Plant Pathol. 2022 Dec;23(12):1737-1750. doi: 10.1111/mpp.13255. Epub 2022 Sep 12. PMID: 36094814; PMCID: PMC9644278.
UHPLC
Chromatographic separation of SPLFA and SPLF was done on an Agilent 1290 UHPLC system coupled to an auto sampler, a diode array detector (DAD) and an ACQUITY UHPLC? HSS T3 column (2.1 ×100 mm, 1.8 μm; Waters, Milford, MA, USA)maintained at 30 ℃. Eluent A was 0.05% formic acid in water and eluent B was 0.05% formic acid in acetonitrile. Elution of the SPLPA compounds was achieved using the following linear gradient elution (in %B): 0 min, 5%; 20 min, 60%; 20.1 min, 5%; 30 min, 5%, and for the SPLF compounds: 0 min, 20%, 25 min, 50%, 25.1 min, 20%, 35 min, 20%. The injection volume was 5 μL, with a flow rate of 0.3 mL/ min. All sample solutions were filtered through a 0.22 μm PTFE filter (Agilent Technologies) before analysis. The detection wavelength was 320 nm for SPLPA and 254 nm for SPLF.
References：
[1] Luo D , Mu T , Sun H .Profiling of phenolic acids and flavonoids in sweet potato (Ipomoea batatas L.) leaves and evaluation of their anti-oxidant and hypoglycemic activities[J].Food Bioscience, 2020, 39(44):100801.DOI:10.1016/j.fbio.2020.100801.
HPLC-DAD
The identification and quantification of phenolics in the S. miltiorrhiza leaves extract before and after enrichment were analyzed using an Agilent 1260 Infinity LC system (Agilent Co., Palo Alto, CA, USA).10 μL of sample dissolved in 80 % methanol was injected into the HPLC system, and phenolics were chromatographed over a YMC-Pack ODS-A (250 × 4.6 mm, 5 μm) column at room temperature with a combination of 0.1 % trifluoroacetic acid (A) and acetonitrile (B) at a flow rate of 1 mL/min. The procedure for elution was as follows: 0? 5 min, 10 % B; 5? 10 min, 10 %–12 % B; 10? 20 min, 12 %–15 % B; 20? 25 min, 15 %–20 % B; 25? 60 min, 20 %–30 % B; 60? 65 min, 30 %–100 % B; 65? 70 min, 100 % B; 70? 80 min, 100 %-10 % B. Phenolics were detected by a diode array detector (DAD) at 280 nm.
References：
[1] Hou M , Hu W , Hao K ,et al.Enhancing the potential exploitation of Salvia miltiorrhiza Bunge: Extraction, enrichment and HPLC-DAD analysis of bioactive phenolics from its leaves[J].Industrial Crops and Products, 2020, 158:113019.DOI:10.1016/j.indcrop.2020.113019.
UV
Extraction and determination of total flavonoids A total of 5 g of fresh V. amygdalina leaves were ground into powder and extracted by 70 mL 95% ethanol for 2 h. After filtration, liposoluble substances were removed by solvent extraction with 35 mL petroleum ether. The above 10 mL extracted lipid was added into a 25 mL volumetric flask. The 2.5 mL 30% ethanol and 0.75 mL 5% sodium nitrite solution were added into volumetric flask, mixing and standing for 5 min. Then, 0.75 mL10% aluminum nitrate solution was added, mixing and standing for 5 min. Subsequently, 10 mL 1 mol/L NaOH solution was added and agitated. Last, dilute with 30% ethanol to volume, incubating for 10 min. Three biological repetitions were performed. The 10 mL extracted lipid was added into a 25 mL volumetric flask, and rutin (Solarbio HPLC >98%) was used as standard for manufacturing a standard curve. Ultraviolet spectrophotometer was used to set the wavelength at 510 nm to measure the absorbance.
References：
Shui L, Huo K, Chen Y, Zhang Z, Li Y, Niu J. Integrated metabolome and transcriptome revealed the flavonoid biosynthetic pathway in developing Vernonia amygdalina leaves. PeerJ. 2021 Apr 26;9:e11239. doi: 10.7717/peerj.11239. PMID: 33981500; PMCID: PMC8083182.
UPLC-QTOT-MS
The C18 (2.1 × 100 mm, 1.7 μm) was kept at 30 ℃. The injection volume was 1 μL, and the elution was completed in 18 min with a flow rate of 0.3 mL/min. Solvents A (water + 0.1% formic acid) and B (acetonitrile) were used in the following gradients: 0–2 min (5–10% B), 2–10 min (10–20% B), 10–15 min (20–40% B), 15–17 min (40–70% B), and 17–18 min (70–100% B). The PDA spectra for phenolic compounds were measured at 320 nm and 350 nm. For MS analysis, anthocyanins were analyzed in the positive ion (PI) mode; other phenolic compounds were analyzed in the negative ion (NI) modes. The MS parameters were as follows: source temperature of 120 ℃, desolvation temperature of 250 ℃(400℃ in the positive ion), cone gas flow 50 L/h, desolvation gas flow 600 L/h (800 L/h in the positive ion), source capillary of 3.0 kV. The MS analysis was performed using mass scanning from m/z 50 to 1,500.
References：
[1] Xiang Z , Lin C , Zhu Y ,et al.Phytochemical profiling of antioxidative polyphenols and anthocyanins in the wild plant Campanumoea lancifolia (Roxb.) Merr[J].International Journal of Food Properties, 2021, 24(1):105-114.DOI:10.1080/10942912.2020.1867570.
