Page 178 - EJMO-9-3

Eurasian Journal of
Medicine and Oncology Research on DSY in treating gastritis

Shanghai Yuanye Biotechnology (China)–were used as a Hypersil GOLD column (2.1 mm × 100 mm, 1.8 μm)
reference compounds, each with a purity exceeding 98%. maintained at 40℃. The binary mobile phase consisted
The Bradford protein concentration kit was obtained from of 0.1% formic acid in water (A) and acetonitrile (B).
Beyotime Biotechnology (China). 1-Phenyl-3-methyl-5- The gradient elution procedure was as follows: 0 – 2 min,
pyrazolone (PMP; P816062) was purchased from Shanghai 10 – 15% B; 2 – 3 min, 15 – 26% B; 3 – 6 min, 26 – 28%);
Macklin Biochemical Technology (China). Trifluoroacetic 6 – 8 min, 28 – 28% B; 8 – 9 min, 28 – 75% B; 9 – 11 min,
acid was purchased from Shanghai Yien Chemical 75 – 75% B; 11 – 15 min, 75 – 80% B; 15 – 17 min, 80 – 90%
Technology (China). Ammonium acetate was obtained B; 17 – 19 min, 95% B. The injection volume was 2 μL.
from Sigma-Aldrich (USA). Mass spectrometric detection was performed using an
Orbitrap Exploris 120 (Thermo Fisher Scientific Inc.,
2.2. Preparation of DSY decoction USA) in both positive and negative ion scanning modes.
DSY is composed of DS, TX, and SR in a 10:1:1 ratio. Data acquisition was conducted using Full MS/dd-MS2
Initially, DS was extracted using ten times its weight in scanning and a predefined DSY precursor ion list. The “if
water by refluxing and boiling for 2 h at 100°C. TX and idle pick others” function was turned on.
SR were then added to the DS residue and extracted using The precursor ion list was constructed using constituent
eight times the weight of water over low heat for 1 h. The information on DS, TX, and SR obtained from databases
two extracts were combined, concentrated, and dried. such as CNKI, TCMSP, and Google Scholar. The Xcalibur
Individual extracts of DS, TX, and SR were prepared using 4.2 software (Thermo Fisher Scientific Inc., USA) was
the same method. used to extract information on potential compounds and
2.3. HS-GC-MS analysis of DSY decoction identification by comparison against an in-house database
and reference compounds.
Lyophilized powders of the aqueous extract of DSY,
DS, TX, and SR (all passed through a 50-mesh sieve) 2.5. Characterization of DSY polysaccharides
were accurately weighed for analysis. The equilibration Lyophilized powders of DSY, DS, TX, and SR (10 g each)
temperature was set at 120°C. The injection port, transfer were dissolved in 5 mL of distilled water. Then, 20 mL of
line, and detector temperatures were set to 130°C, 140°C, ethanol was added to the solution. The suspension was
and 220°C, respectively. The equilibration time was 15 min, incubated at 4°C for 48 h. The precipitated underlayer was
injection time 1 min, and pressure equilibration time dissolved in distilled water and deproteinized using the
1 min. An HP-5 MS elastic quartz capillary column (30 m Sevag method. The crude polysaccharide samples were
18
× 0.25 mm, 0.25 μm; Agilent Technologies Inc., USA) then lyophilized.
was used to separate sample, with high-purity helium as
the carrier gas. The pre-column pressure was 9.8 psi, and Total sugar content was measured using the phenol-
19
the column flow rate was 1.2 mL/min. Split injection was sulfuric acid colorimetric method with D-glucose as
performed at a 1:20 ratio. The column heating program is the standard at 490 nm. Total uronic acid content was
provided in Table S2. measured using the m-hydroxyphenyl colorimetric
method with galacturonic acid as the standard. Protein
20
The Agilent MassHunter qualitative analysis navigator content was evaluated using the Coomassie Brilliant Blue
B.08.00 (Agilent Technologies Inc., USA) equipped with assay with bovine serum albumin as the standard. 21
the NIST.17 standard spectral library was utilized for The monosaccharide composition of the samples
compound identification. Only compounds with both was analyzed by HPLC with pre-column derivatization.
a positive match and reverse match score >80% were
retained. The relative abundance of each volatile compound Polysaccharide samples were hydrolyzed with trifluoroacetic
was determined using peak area normalization. acid (TFA) for 6 h at 105°C. Subsequently, 200 μL each of
the acid-hydrolyzed sample and monosaccharide standard
2.4. UHPLC-Orbitrap Exploris MS analysis mixture were accurately aliquoted. The hydrolysates and
monosaccharide standard mixture were converted to their
Sample powders (DSY, DS, TX, and SR) and 19 reference PMP derivatives according to the previous method with
compounds were dissolved in 80% methanol. The mixture some modifications The sample and reference standard
22
was centrifuged at 12,000 rpm for 10 min at 4°C and the samples were analyzed using HPLC at 250 nm at 30℃.,
process was repeated once. The resulting supernatant was using a mobile phase of 17% organic phase (acetonitrile)
used for analysis.
and 83% aqueous phase (0.05 M ammonium acetate).
UHPLC separation was performed on a Vanquish The same procedure was used to analyze reference
UHPLC System (Thermo Fisher Scientific, USA) using monosaccharides. The crude DSY polysaccharide powders

Volume 9 Issue 3 (2025) 170 doi: 10.36922/EJMO025160124