material

Chinese herbal medicines and standards Gallnut, rhubarb, and skullcap were purchased from
Sichuan Provincial Institute of Traditional Chinese Medicine; Gallic acid, 1,8-
dihydroxyanthraquinone, emodin, aloe emodin, baicalin, quercetin and other standards were
purchased from the China Institute for the Control of Pharmaceutical and Biopharmaceutical
Properties.

The test strain Flavobacterium columnare was purchased from the Institute of Hydrobiology,
Chinese Academy of Sciences; Aeromonas sobria, A. caviae, and Edwardsieda tarda were
isolated and identified by the Department of Microbiology, Rongchang Campus, Southwest
University.

Culture medium Beef paste (5 g), sodium hydroxide (5 g) and egg peptone (l0 g) were used to
prepare ordinary medium for the culture of Aeromonas mild, Aeromonas guinea pigs and
Edwards retardi. Pancreatic peptone medium was prepared with pancreatic peptone (0.5 g),
sodium acetate (0.2 g), beef extract powder (0.2 g) and yeast paste (0.5 g) for the culture of
Flavobacterium columnar. Boil the drug, fully dissolve the components, cool to room
temperature, and adjust the pH to 7.6-7.8 with a certain concentration of sodium hydroxide and
hydrochloric acid. The solid medium was sterilized at 115°C for 30min with 1.5% agar.

1.2 Method

Preparation of sample liquid Preparation of total active ingredient sample liquid: Weigh 2 g of
compound gallnut antibacterial agent coarse powder and place it in a Soxhlet extractor, add 16
mL of 70% ethanol, adjust the pH to 2 with hydrochloric acid, and reflux and extract at 76°C for
90 minutes , extract 3 times, combine the extracts, evaporate until there is no alcohol smell, add
water to the filtrate to 100 mL, shake well, take out, extract 3 times with anhydrous ether at
room temperature, combine the extracts, and evaporate the ether in a ventilated place, leaving
the remaining crystalline material Dissolve with absolute ethanol to 10 mL, which is the sample
solution. The preparation of single prescription gallnut, rhubarb and scutellaria baicalensis
sample liquid is the same as the compound prescription.

Preparation of sample solutions of each monomer component: Use the compound sample
solution prepared by the above method to obtain each monomer component through column
chromatography, combine the relevant components, evaporate the solvent in a water bath to
obtain each crystalline monomer, then dissolve it with distilled water, and combine the relevant
components Distilled water was added to dissolve to 10 mL to obtain anthraquinone sample
solution (including 1,8-dihydroxyanthraquinone, emodin, and aloe-emodin), baicalin sample
solution, quercetin sample solution, and gallic acid sample solution. Tannin sample liquid.
Sterilize at high temperature and store in a refrigerator at 4°C for later use.
Preparation of bacterial liquid: Inoculate Aeromonas temperate, Aeromonas guinea pig and
Edwardsiella tarda in ordinary liquid culture medium, and inoculate Flavobacterium columnaris
in tryptone liquid culture medium. Place the inoculated petri dishes in a 35°C incubator for 24
hours. Use sterile 0.9% sodium chloride solution to prepare a bacterial solution of 150
million/mL by turbidimetric method and store it at low temperaturespare.

Thin-layer analysis of the active ingredients of the compound: Take each single sample solution
for thin-layer analysis, and compare the R value of the compound sample to determine whether
new substances are produced and disappeared. Take the compound extract for thin layer
analysis, and compare the R values of each sample point in the compound with the standard to
determine the sample composition.

Preparation of thin plates: Use a 10 cm × 20 cm glass plate for thin layer analysis, soak it in
lotion, wash it, dry it, lay it on the 939 fully automatic thin layer plate maker, and wipe it with
95% ethanol before use. . Weigh 15 g of silica gel GF254, put it into a mortar, add an appropriate
amount of 0.5% sodium carboxymethylcellulose, and grind it evenly. Make it into a thin paste
and lay it on a thin-layer plate maker with a thickness of 0.3 mm. Let it dry and then Activate at
110℃ for 0.5h and put in a desiccator for later use.

Spotting: Determine the spotting points on a parallel line 2 cm from the bottom, about 1.5 cm
apart, and spot the samples in sequence from left to right. The microspotter lightly touches the
surface of the silica gel plate, and the diameter of the spots formed does not exceed 2-3 mm.
After spotting, the sample is naturally air-dried.

Chromatography: 9 developing agents were combined with different drugs and proportions of
benzene, ethanol, formic acid, ethyl acetate, and pyridine, and finally the thin layer developing
agent with the best effect was selected, that is, benzene: ethanol: formic acid (7: 3: 1 ). Add 20
mL of developing agent to the development cylinder and shake well. Paste filter paper soaked in
chromatography agent on the four walls of the chromatography cylinder to saturate the
chromatography cylinder. Place the end of the sheet with the sample into the chromatography
cylinder, tilt it slightly, cover the chromatography lid, and develop the layer upward at room
temperature. When the spreading agent is 1-2 cm away from the front edge of the sheet, stop
chromatography, take out the sheet, mark the front edge of the spreading layer, and let it air
dry naturally.

Color development: Ammonia fumigation-ferric chloride color development. Spray the thin layer
plate with concentrated ammonia solution, then spray the thin layer plate with 1% ferric
chloride ethanol solution, develop color under sunlight, and measure the relative shift value Rf
of each component point after color development.

Column chromatography separates the active ingredients of the compound. Sample

preparation: Take 2.0 g of the compound powder and prepare it with the same method as
above. Keep the crystallized sample for later use. Packing the column: Pack the column by dry
method. Take about 30 g of column chromatography silica gel and put it into the column
chromatography tube using a funnel. At the same time, tap the column wall continuously to
make the column evenly packed and set aside.

Sample loading: Take the prepared dry sample, add 2.0 g of column chromatography silica gel,
dissolve it with the prepared eluent, and then put it into an oven to dry. Use a funnel to put the
dried sample silica gel into the column chromatography tube, and place it in the column
chromatography tube. Stuff a ball of cotton on top to prevent the sample from floating.

Elution: Use the thin-layer developing agent selected previously, that is, benzene: ethanol:
formic acid (7: 3: 1) as the eluent.

Collection: Collect with an automatic collector, collect a sample every one minute, and use TLC
to track and detect, and combine the eluates of the same spots at the same time.

In vitro antibacterial test: Antibacterial test of each component: Use a sterile cotton swab to
evenly spread Aeromonas temperarum, Aeromonas guinea pig and Edwardsiella tarda with a
concentration of 150 million/mL on the ordinary solid medium, and Column-shaped Flexibacter
was spread on the solid tryptone culture medium, and then a hole was made in a sterile round
glass tube with a diameter of 6 mm, and 30 μL of the drug solution was drawn with a
micropipette and injected into the hole. Then place the petri dish in a 37°C incubator for 24
hours, take it out for observation, and use a caliper to measure the diameter of the inhibition
zone. Take the average of 3 repetitions.

Combined drug susceptibility test: Use a sterile cotton swab to evenly apply Aeromonas mildum
solution with a concentration of 150 million/mL on the ordinary solid culture medium, and then
use a sterile round glass tube with a diameter of 6 mm to punch a hole in the solid medium.
Punch holes side by side in the culture medium, use a micropipette to draw 30 μL of the
corresponding drug solution of each component and inject it into the holes. Then place the petri
dish in a 37°C incubator for 24 hours, then take it out and observe the shape of the junction of
the antibacterial rings of the two components after combined treatment. The straight
antibacterial junction area of two drugs indicates synergistic effect, the obtuse angle of the
antibacterial junction area of two drugs indicates additive effect, the sharp antibacterial junction
angle of two drugs indicates irrelevant effect, and the cut-shaped junction of the antibacterial
zone of two drugs indicates antagonistic effect.

2 results

2.1 Thin layer analysis of active ingredients

As shown in Figure 1, it can be seen from each single sample point that there are three main
components in gallnuts, rhubarb also has three components, while only one component of
skullcap appears, and all single ingredients can be found in the compound. Ingredient points.
There are no new ingredients in the compound ingredients except those expressed in the single
recipe, which shows that this extraction process can well extract the ingredients in the
compound, and the ingredients analyzed in the thin layer analysis spectrum can represent the
main ingredients of the compound.

As shown in Figure 2, in the thin-layer analysis of the standard and compound sample solutions,
the components can be separated well, and each spot is clear. The R results corresponding to
each component point of the standard and the compound are consistent, which is consistent
with the component judgment in TLC analysis. standard. Arranged by R value, they are tannins,
gallic acid, quercetin, baicalin, aloe-emodin, emodin and 1,8-dihydroxyanthraquinone. And it
can be seen from the color depth and size of the spots that gallic acid contains the largest
amount.

2.2 Separation and identification of active ingredients of the compound by column

chromatography

The active ingredients of the compound gallnut antibacterial agent are separated by silica gel
column chromatography. Benzene: ethanol: formic acid (7: 3: 1) is used as the eluent, which can
separate the components well. An automatic collector was used to collect a sample every 1 min,
followed by TLC tracking, and a total of 93 samples were collected (Figure 3). A-G are standard
products. By comparison, it can be seen that samples 1-11 are 1,8-di-hydroxyanthraquinone, 12-
15 are emodin, 17-21 are aloe-emodin, 24-30 are baicalin, and 33-47 are quercetin. , 49-77 are
gallic acid, 80-93 are tannins. Comparing the collected amounts, gallic acid is the most
abundant, which is consistent with the content reflected by the spot size of each component in
compound thin-layer analysis.