D. Jia, et al.
Bioorganic&MedicinalChemistry28(2020)115697
distilled water to neutral pH. After washed with petroleum ether,
neutral crystals were evaporated in vacuum at 80 °C (5.38 g). With this
method, BBH was obtained with a 53% yield.
Table 1
Disesase activity index scoring system.
Score
Weight loss (%)
Fecal bleeding
Stool consistency
Normal
0
1
2
3
4
0–2
None
2.2.3.1. HPLC analysis. HPLC was used to monitor the synthesis
reaction of BBH. Samples of baicalin, compound X4 and BBH were
analyzed using YMC - Pack ODS – AQ (250 mm × 4.6 mm, 5 μm) at 30
°C. The mobile phases consisted of acetonitrile (45%) and 0.2% formic
acid aqueous solution (55%). The flow rate was set at 1.0 ml/min. The
sample injection volume was 20 µL. Purity of BBH was also detected
using HPLC under the same conditions.
> 2–5
–
> 5–10
> 10–15
> 15–20
Hemoccult +
–
Soft
Visible bleeding
liquid
berberine - treated group (50 mg/kg) and the BBH - treated group
(50 mg/kg). DSS was dissolved in pure water to obtain a 5% DSS so-
lution. To develop mouse model of UC, mice in model and treatment
groups were allowed free access to 5% DSS solution as drinking water
for 7 days, the solution was freshly prepared. UC mice started to receive
treatments from day 1 and lasted for 14 days. Dose of berberine was set
according to previous study,30 while the concentrations of baicalin and
BBH were in line with berberine. Mice in control and UC model groups
were administered with pure water.
2.2.3.2. BBH. BBH, yellow solid; UV: λmax 205.2, 217.7, 273.4, 333.4,
and 440.7 nm; 1H NMR (CDCl3, 300 MHz) δ: 7.76 (1H, s, H - 1), 7.09
(1H, s, H - 4), 3.42 (2H, t, J = 5.8, H - 5), 4.85 (2H, t, J = 5.8, H - 6),
9.31 (1H, s, H - 8), 7.96 (1H, d, J = 9.1, H - 11), 8.00 (1H, d, J = 9.1, H
- 12), 8.81 (1H, s, H - 13), 6.18 (2H, s, H - 15), 3.76 (3H, s, H - 16), 4.45
(2H, t, J = 6.3 Hz, H - 20), 1.80 (2H, m, H - 18), 1.93 (2H, m, H - 19),
4.22 (2H, t, J = 18.6 Hz, H - 17), 12.39 (1H, s, 5′ - OH), 8.50 (1H, s, 6′ -
OH), 8.05 (2H, d, J = 4.5 Hz, 2″, 6″), 7.54–7.57 (2H, m, 3″, 5″), 7.59
(1H, m, 4″), 7.09 (1H, s, 3′), 6.90 (1H, s, 8′), 5.40–5.59 (3H, m,
2′″,3′″,4′″ - OH), 5.26 (1H, d, J = 4.2 Hz, 1′″), 4.44 (1H, d, J = 6.3 Hz,
5′″) and 3.16–3.33 (3H, m, 2′″, 3′″, 4′″); 13C NMR (CDCl3, 300 MHz) δ:
105.50 (C - 1), 149.67 (C - 2), 148.72 (C - 3), 108.25 (C - 4), 130.49 (C -
4a), 26.25 (C - 5), 56.42 (C - 6), 144.28 (C - 8), 137.20 (C - 8a), 120.04
(C - 9), 147.50 (C - 10), 120.37 (C - 11), 122.65 (C - 12), 132.66 (C -
12a), 125.93 (C - 13), 142.05 (C - 14), 120.76 (C - 14a), 101.99 (C - 15),
55.25 (C - 16), 73.16 (C - 17), 24.28 (C - 18), 25.91 (C - 19), 64.59 (C -
20), 162.96 (C - 2′), 104.27 (C - 3′), 182.16 (C - 4′), 149.25 (C - 5′),
131.93 (C - 6′), 150.62 (C - 7′), 93.24 (C - 8′), 146.33 (C - 9′), 105.69 (C
- 10′), 130.49 (C - 1″), 126.03 (C - 2″, 6″), 128.96 (C - 3″, 5″), 130.04 (C
2.4.3. Disease activity index (DAI)
Both weight loss and appearance of fecal bleeding were recorded
every day. Severity of ulcerative colitis was assessed using DAI. The
score distribution was adapted from previously study with slight
modification as shown in Table 1.31 Occult blood in the feces was
measured using Hemoccult kit.
2.4.4. Tissue collection and processing
After 14 days treatments, mice were sacrificed. Entire colon were
collected, the length and weight were measured. Organs of spleen and
cecum were removed and weighed.
- 4″), 99.71 (C - 1′″), 72.68 (C - 2′″), 74.94 (C - 3′″), 71.14 (C - 4′″),
+
2.4.5. Colonic damage
75.20 (C - 4′″) and 168.99 (C - 4′″); ESI - MS: m/z, [C44H40NO15
]
,
Fat and mesentery of colon were cleaned on ice - cold plate. Colon
mage according to the criteria described in Table 2.32 After macroscopic
observation, one cm of the distal colon was cut and fixed in PBS buf-
fered 10% formalin for 24 h. Distal colon were then embedded in par-
affin, cut into 5 μm thick sections and stained with H & E. Histological
scoring (HS) was given to each microscopic field according to the his-
tological disease scoring system (Table 2), the number of fields per
section were large than 12.
822.24.
2.3. In vitro antibacterial test
Baicalin, berberine and BBH were prepared and diluted with culture
medium accordant with ratios of 1:2.5, 1:5, 1:10, 1:20, 1:40, 1:80,
1:160, 1:320 and 1:640. These drug mediums were then sterilized using
circulating steam. With pure water as the control, the minimal in-
hibitory concentrations (MIC) of treatments against eight kinds of
common pathogenic microorganisms were tested through micro - di-
lution method. Four to five colonies were picked from purified strains,
inoculated to the nutrient broth medium, and cultured in an incubator
at 37 °C for 8 h. Afterwards, it was diluted to the liquid containing
1.5 × 105 CFU/mL bacteria. In sterilized 96 - well polystyrene micro -
well plate, 0.1 ml of each strain liquid were added to holes containing
different concentrations of treatment agents or pure water. After in-
cubated at 37 °C for 24 h, the results were observed.
2.4.6. Activities assay of SOD and MPO
Activities of SOD and MPO in colon were measured using related
biochemistry assay kits. SOD activity was assayed by xanthine oxidase
method while MPO was measured by colorimetric method. The absor-
bance was determined at 550 nm by using microplate reader.
2.4.7. Real - time quantitative PCR (RT - qPCR)
Total RNA was extracted from colonic tissue using TRIzoI reagent,
followed by first strand cDNA synthesis. RNA quantification were per-
formed using the SYBR Premix Ex Taq@ with BIORAD CFX96 (BIO -
RAD, USA). All RT - qPCR reactions were carried out in triplicate.
GAPDH house keeping gene was used as a reference standard. Relative
expression levels of the target genes were calculated by 2−ΔΔCt method.
For mRNA expression, data were first normalized relative to the ex-
pression of GAPDH, and then relative to the model group. Primers used
in real - time quantitative PCR were shown in Table 3.
2.4. In vivo experiment
2.4.1. Mice
Specific pathogen - free (SPF) male BALB/c mice (18–22 g) were
purchased from Experimental Animal Center of Guangdong [SCXK
(Guangdong) 2013-0034]. Mice were housed in SPF condition under a
12 h light - dark cycle with ad libitum access to water and food. All
experiments and procedures were carried out according to the
Regulations of Experimental Animal Administration issued by the State
Committee of Science and Technology of China. Every effort was made
to minimize the number of animals used and their suffering.
2.4.8. Western blotting (WB)
Total protein of colon was extracted and fractionated by sodium
dodecyl sulfate polyacrylamide gel electrophoresis and transferred to
polyvinylidene difluoride membranes. Then, the membranes were
blocked with 5% nonfat milk in Tris - buffered saline with Tween - 20
(TBST) for 1 h at room temperature. Membranes were incubated with
2.4.2. DSS - induced ulcerative colitis and treatments
In present study, mice were randomly divided into 5 groups (n = 8):
control group; UC model group; baicalin - treated group (50 mg/kg);
3
Jia, Dan
