CN112603936B - Composition for inhibiting aeromonas, preparation method and application thereof - Google Patents

Abstract

The invention provides a composition for inhibiting Aeromonas, a preparation method and application thereof. Serve evenly. The composition for inhibiting Aeromonas according to the present invention contains doxycycline, extracts of Scutellaria chinensis and extracts of Radix chinensis, and the combination of the three can significantly inhibit the bacteria of the genus Aeromonas The composition can be used for the preparation of medicaments and feed additives.

Description

A kind of composition for inhibiting Aeromonas and its preparation method and application

technical field

The invention belongs to the field of aquatic products, and in particular relates to a composition for inhibiting Aeromonas, a preparation method and application thereof.

Background technique

my country is a big country in aquaculture, and the aquaculture density and output of aquatic products are high. However, due to the backward aquaculture technology and facilities, it often leads to the frequent occurrence of bacterial diseases in fish. At present, antibiotic-based drug control is still the most effective and direct method in aquaculture production, and plays a very important role in the prevention and control of fish bacterial diseases. However, the long-term use of antibiotics in production practice will lead to the emergence of resistant strains in a short period of time.

SUMMARY OF THE INVENTION

In view of this, the present invention aims to overcome the defects in the prior art, and proposes a composition for inhibiting Aeromonas and its preparation method and application.

In order to achieve the above object, the technical scheme of the present invention is achieved in this way:

A composition for inhibiting Aeromonas, the composition is made from the raw materials comprising the following parts by weight:

Doxycycline 0.001-0.1 servings,

10-100 servings of Sophora japonica Extract,

Qianjinpu extract 10-100 servings.

Preferably, the composition is made from the raw materials comprising the following parts by weight:

Doxycycline 0.001-0.05 servings,

10-50 servings of Sophora japonica extract,

Qianjinpu extract 50-100 servings.

Preferably, the composition is made from the raw materials comprising the following parts by weight:

Doxycycline 0.008-0.028 servings,

32-50 servings of Sophora japonica Extract,

Qianjinpu extract 85-100 servings.

Further, the extract of Qianjinba is prepared by including the following steps: after pulverizing and sieving the Qianjinba powder, the Qianjinba powder is added to 10-30 times the concentration of 60-70%. In ethanol, leaching for 1-2 hours, the obtained leaching solution is refluxed and extracted at 80-90° C., and then concentrated to obtain the extract of Qianjinpu.

The preparation method of the composition for inhibiting the genus Aeromonas includes the following steps: uniformly mixing the extract of Radix Glycyrrhizae and the extract of Qianjinpu, and then adding doxycycline to the mixture and mixing uniformly.

A kind of preparation method of Fructus Heliconiae extract, comprising the steps:

(1) after pulverizing and sieving the Scutellaria chinensis powder, the obtained powder is heated at 60-90 ° C for 5-30 minutes;

(2) then the powder after heating is divided into three parts of the same quality of Fructus chinensis powder, the first part of Fructus chinensis powder is added to 10-30 times the amount of ethanol with a concentration of 40-55%, and the extraction is carried out for 12 -24 hours to obtain the extract A, add the second part of the powder of Scutellaria officinalis to 10-30 times the amount of ethanol with a concentration of 60-75%, and extract for 12-24 hours to obtain the extract B. The third part of the Radix Glycyrrhizae powder is added to 10-30 times the amount of ethanol with a concentration of 80-95%, and the extract is extracted for 24-48 hours to obtain the extract C;

(3) The leaching solution A, the leaching solution B and the leaching solution C are mixed uniformly and filtered, and the obtained filtrate is obtained after rotary steaming.

Further, the temperature of the heating step in the described step (1) is 60-80 ° C, and the time is 15-30 minutes; In the 55% ethanol, the second part of the Radix Glycyrrhiza powder is added to the ethanol whose concentration is 60-65%, and the third part of the Radix Glycyrrhiza powder is added into the ethanol whose concentration is 80-90%.

An extract of Phyllostachys chinensis, the extract is prepared by the method.

Application of a composition for inhibiting Aeromonas in preparing a medicine for inhibiting Aeromonas.

Further, the composition comprises doxycycline, Scutellaria chinensis extract and Qianjinpu extract.

Compared with the prior art, the present invention has the following advantages:

The composition for inhibiting Aeromonas according to the present invention contains doxycycline, Scutellaria chinensis extract and extract of Qianjinpu, and the combination of the three has obvious inhibition on Aeromonas bacteria The composition can be used for the preparation of medicaments and feed additives.

Detailed ways

Unless otherwise defined, technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples are conventional biochemical reagents unless otherwise specified; the experimental methods are conventional methods unless otherwise specified.

The present invention will be described in detail below with reference to the embodiments.

The Aeromonas Schubert strains, Aeromonas hydrophila, and Aeromonas pipiens described in the embodiments of the present invention were donated by the Aquatic Diseases and Immunity Laboratory of the Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences.

Example 1 Preparation of Scutellaria chinensis extract

After smashing and sieving the Scutellaria japonica powder, accurately weigh 600g of the powder and heat it at 80°C for 20 minutes; then divide the heated powder into three parts of the same quality of Scutellaria japonica powder. The blood vine powder was added to 20 times the amount of ethanol with a concentration of 50%, and extracted for 12 hours to obtain an extract A. The second part of the vine root powder was added to 20 times the amount of ethanol with a concentration of 60%. After leaching for 12 hours, the extraction solution B was obtained, and the third part of the powder was added to 20 times the amount of ethanol with a concentration of 90%, and the extraction was performed for 48 hours to obtain the extraction solution C; The solution A, the extraction solution B and the extraction solution C are mixed evenly and then filtered, and the obtained filtrate is rotary-evaporated to obtain the extract A of S. chinensis.

After smashing and sieving the Scutellaria japonica powder, accurately weigh 600g of the powder and heat it at 50°C for 20 minutes; then divide the heated powder into three parts of the same quality of Scutellaria officinalis powder. The blood vine powder was added to 20 times the amount of ethanol with a concentration of 50%, and extracted for 12 hours to obtain an extract A. The second part of the vine root powder was added to 20 times the amount of ethanol with a concentration of 60%. After leaching for 12 hours, the extraction solution B was obtained, and the third part of the powder was added to 20 times the amount of ethanol with a concentration of 90%, and the extraction was performed for 48 hours to obtain the extraction solution C; The solution A, the extraction solution B and the extraction solution C are mixed evenly and then filtered, and the obtained filtrate is rotary-evaporated to obtain the extract B of Scutellaria chinensis.

After smashing and sieving the Scutellaria japonica powder, accurately weigh 600g of the powder and heat it at 50°C for 20 minutes; then divide the heated powder into three parts of the same quality of Scutellaria officinalis powder. The blood vine powder was added to 20 times the concentration of ethanol with a concentration of 35%, and extracted for 12 hours to obtain the extract A. The second part of the blood vine powder was added to 20 times the amount of ethanol with a concentration of 50%. After leaching for 12 hours, the extract B was obtained. The third part of the powder was added to 20 times the amount of ethanol with a concentration of 70%, and the extract was extracted for 48 hours to obtain the extract C; The solution A, the extraction solution B and the extraction solution C are mixed evenly and then filtered, and the obtained filtrate is rotary evaporated to obtain the Scutellaria chinensis extract C.

After smashing and sieving the Scutellaria japonica powder, accurately weigh 1500g of the powder and heat it at 50 ° C for 20 minutes; The blood vine powder was added to 20 times the amount of ethanol with a concentration of 60%, and extracted for 12 hours to obtain an extract A. The second part of the vine blood vine powder was added to 20 times the amount of ethanol with a concentration of 80%. Extraction for 12 hours to obtain extraction solution B, add the third part of the Fructus chinensis powder into 20 times the amount of ethanol with a concentration of 95%, and extract for 48 hours to obtain extraction solution C; The solution A, the extraction solution B and the extraction solution C are mixed evenly and then filtered, and the obtained filtrate is rotary evaporated to obtain the extract D of Scutellaria chinensis.

After crushing and sieving Qianjinpu, get Qianjinpu powder, accurately weigh 1500g Qianjinpu powder and add it to 20 times the amount of ethanol with a concentration of 70%, extract for 2 hours, and reflux the obtained extract at 80 ° C for extraction , and then concentrated to obtain the extract of Qianjinpu.

Mix 48 g of the extract A of Scutellaria officinalis and 90 g of the extract of Qianjinpu, and then add 20 mg of doxycycline to the mixture to obtain the composition A after mixing evenly.

Mix 48 g of the extract B of Scutellaria officinalis and 90 g of the extract of Qianjinba, and then add 20 mg of doxycycline to the mixture to obtain the composition B after mixing evenly.

Mix 48 g of Scutellaria officinalis extract C and 90 g of extract of Qianjinba, and then add 20 mg of doxycycline to the mixture and mix evenly to obtain composition C.

Mix 48 g of the extract D of Scutellaria officinalis and 90 g of the extract of Qianjinpu, and then add 20 mg of doxycycline to the mixture to obtain the composition D after mixing evenly.

Accurately weigh 90 g of the extract of Qianjinpu, and then add 20 mg of doxycycline to it and mix it evenly to obtain the composition E.

Example 2 The antibacterial effect of the extract of Gallatia chinensis on Aeromonas hydrophila

1. Preparation of culture medium

BHI medium was purchased from BD Company in the United States; 5% sheep blood agar plates, bacterial biochemical micro-identification tubes, etc. were purchased from Guangzhou Huankai Microorganism Co., Ltd.

2. Preparation of bacterial suspension

Test bacteria culture: The Aeromonas hydrophila strain was activated, streaked on the solid medium with an inoculating needle, and after culturing at 30°C for 24 hours, a single colony was picked and inoculated on the nutrient broth medium for 24 hours with shaking. The bacterial solution was diluted to an appropriate concentration by the plate counting method, and the concentration of the bacterial solution was 1×108 CFU/mL.

3. Drug susceptibility test

The drug susceptibility test adopts the agar diffusion method, and the bacterial suspension cultured to the logarithmic growth phase is diluted to a concentration of about 1 × 103 CFU/mL, and a sterile cotton swab is used to dip the bacterial solution and spread it evenly on the medium. Punch holes evenly on the plate, punch 4 holes on each plate, and make a mark. Add 100 μl (1 g/mL) of the composition to each well, and then put it into a 30°C incubator for 24 hours; after taking out the plate, observe whether there is an inhibition zone around the well, and if so, measure the diameter of the inhibition zone with a vernier caliper. Three replicates were performed for each drug, while sterile water was used as a blank control. The results were judged according to the standard introduced in "Pharmacology of Traditional Chinese Medicine": the inhibition zone was ≥20 mm, which was considered extremely sensitive; the inhibition zone was 15-19 mm, which belonged to high sensitivity; the inhibition zone was 10-14 mm, which belonged to moderate sensitivity; the inhibition zone was less than 10 mm, which belonged to low sensitivity. Sensitive; no inhibition zone, insensitive (no antibacterial effect).

4. Secondary screening test

Use nutrient broth medium to carry out gradient dilution to the extract by double dilution method, add bacterial suspension to each test tube to make the bacterial concentration reach 102CFU/mL, add composition A, composition B, Composition C, composition E, no composition was used as a blank control, and no bacteria was added as a negative control, each repeated 3 times. Placed in a 28°C shaker with shaking (180 r/min) for 24 hours, and then taken out and compared with the control tube for observation.

Table 1 The diameter of the inhibition zone and the antibacterial grade

project Inhibition zone diameter (mm) Antibacterial grade Composition A 22.94±0.15 +++ Composition B 12.35±0.55 + Composition C 17.26±0.28 ++ Composition D 16.63±0.41 ++ Composition E 9.26±0.42 + blank — — negative control — —

In the above table, "+++" indicates extreme sensitivity, "++" indicates high sensitivity, "+" indicates moderate or low sensitivity, and "-" indicates insensitive (no antibacterial effect).

As can be seen from Table 1, composition A, composition B, composition C, composition D, and composition E all have bacteriostatic effect on Aeromonas hydrophila, wherein the bacteriostatic grade of composition A is extremely sensitive, and the combination The grades of composition C and composition D are high-sensitivity, and the grades of composition B and composition E (without the extract of Phyllostachys chinensis) are moderate or hypoallergenic. The composition has a significant bacteriostatic effect on Aeromonas hydrophila, and the bacteriostatic effect of the composition A is particularly significant, while for the composition B, when preparing the extract of S. Heating activation can not fully extract the bacteriostatic components therein, so that the effect is quite different from that of composition A. For composition C and composition D, ethanol with different concentration gradients is used for leaching, and the effect is Compared with the concentration gradient of ethanol in the preparation composition A, there is also a certain gap, but the effect of different concentration gradients of ethanol on the antibacterial effect of the extract of S. chinensis is much lower than that of the activation temperature. This shows that, in composition A, the combination of the extract of Radix Glycyrrhizae, the extract of Radix chinensis and doxycycline has a significant bacteriostatic effect on Aeromonas hydrophila, and the temperature of heating activation and the concentration of ethanol are changed. The concentration gradient also has a great influence on the bacteriostatic effect of the composition on Aeromonas hydrophila.

Embodiment 3 The influence of the ratio of the extract of Scutellaria officinalis and the extract of Qianjinpu on the antibacterial effect

After smashing and sieving the Scutellaria japonica powder, accurately weigh 600g of the powder and heat it at 80°C for 20 minutes; then divide the heated powder into three parts of the same quality of Scutellaria japonica powder. The blood vine powder was added to 20 times the amount of ethanol with a concentration of 50%, and extracted for 12 hours to obtain an extract A. The second part of the vine root powder was added to 20 times the amount of ethanol with a concentration of 60%. After leaching for 12 hours, the extraction solution B was obtained, and the third part of the powder was added to 20 times the amount of ethanol with a concentration of 90%, and the extraction was performed for 48 hours to obtain the extraction solution C; The solution A, the extraction solution B and the extraction solution C are mixed evenly and then filtered, and the obtained filtrate is rotary-evaporated to obtain the Scutellaria chinensis extract.

After crushing and sieving Qianjinpu, get Qianjinpu powder, accurately weigh 1500g Qianjinpu powder and add it to 20 times the amount of ethanol with a concentration of 70%, extract for 2 hours, and reflux the obtained extract at 80 ° C for extraction , and then concentrated to obtain the extract of Qianjinpu.

48 g of the extract of Scutellaria officinalis was mixed with 90 g of the extract of Qianjinba, and then 20 mg of doxycycline was added to the mixture to obtain the composition A.

5 g of the extract of Scutellaria officinalis and 90 g of the extract of Qianjinpu were mixed uniformly, and then 20 mg of doxycycline was added thereto and mixed uniformly to obtain the composition B.

48 g of the extract of Scutellaria officinalis and 150 g of the extract of Radix chinensis were mixed uniformly, and then 20 mg of doxycycline was added to it and mixed uniformly to obtain the composition C.

5 g of the extract of Scutellaria officinalis and 150 g of the extract of Qianjinpu were mixed uniformly, and then 20 mg of doxycycline was added to the mixture and mixed uniformly to obtain the composition D.

Accurately weigh 90 g of the extract of Qianjinpu, and then add 20 mg of doxycycline to it and mix it evenly to obtain the composition E.

Accurately weigh 48 g of the extract of Scutellaria officinalis, and then add 20 mg of doxycycline to it and mix it uniformly to obtain the composition F.

Accurately weigh 20 mg of doxycycline, dissolve it in 10 times the amount of ethanol with a concentration of 60%, and obtain the composition G after mixing evenly.

Add the bacterial suspension of Aeromonas hydrophila to each test tube to make the bacterial concentration reach 102CFU/mL, and add composition A, composition B, composition C, composition E, composition F to different test tubes , composition G, without the composition as a blank control, without bacteria as a negative control, each repeated 3 times. It was placed in a 30°C shaker with shaking (180 r/min) for 24 hours, and then taken out and compared with the control tube for observation.

Table 2 Diameter of Inhibition Zone and Antibacterial Grade

project Inhibition zone diameter (mm) Antibacterial grade Composition A 22.75±0.18 +++ Composition B 15.82±0.36 ++ Composition C 19.16±0.42 ++ Composition D 12.35±0.22 + Composition E 8.36±0.15 + Composition F 10.84±0.23 + Composition G 7.95±0.55 + blank — — negative control — —

In the above table, "+++" indicates extreme sensitivity, "++" indicates high sensitivity, "+" indicates moderate or low sensitivity, and "-" indicates insensitive (no antibacterial effect).

As can be seen from Table 2, composition A, composition B, composition C, composition D, composition E, composition F and composition G all have bacteriostatic effect on Aeromonas hydrophila, wherein composition A has bacteriostatic effect. The antibacterial grade is extremely sensitive, the grades of composition B and composition C are high sensitivity, the composition D, composition E (without extract of Helicobacter sp.) ciccycline) grades are moderate or hypoallergenic. Thus, it can be seen that the composition containing the extract of Gallatia chinensis and the extract of C. japonicus has a significant bacteriostatic effect on Aeromonas hydrophila, wherein the bacteriostatic effect of the composition A is particularly significant, and the bacteriostatic effect of the composition B is particularly significant. Compared with composition A, there is an obvious gap in effect. The difference between composition B and composition A is that the addition amount of the extract is 5 g. It can be seen that the addition amount of the extract is too low. It will reduce the bacteriostatic effect of the composition; the bacteriostatic effect of the composition C is significantly different from that of the composition A, but the effect is obviously better than that of the composition B; the difference between the composition C and the composition A is that the The addition amount of the extract is 150g. It can be seen that the excessive addition of the extract of Radix chinensis will reduce the bacteriostatic effect of the composition, but the effect of the extract of Radix chinensis on the antibacterial effect of the composition is less than that of the extract of the Radix Glycyrrhizae. thing. The difference between the composition D and the composition A is that the addition amount of the Scutellaria chinensis extract is 5g, and the addition amount of the Radix chinensis extract is 150g, and its bacteriostatic effect is not as good as that of the composition A, the composition B, and the composition C. It shows that the addition amount of the extract of Phyllostachys chinensis and the extract of Phyllostachys chinensis needs to be within a certain range to achieve the optimal antibacterial effect; for the composition E and the composition F, the addition of the extract of Phyllostachys chinensis or the extract of Ph. The composition has a certain bacteriostatic effect on Aeromonas hydrophila, but the bacteriostatic effect is significantly different from the bacteriostatic effect of the composition containing both the extract of Galleria japonica and the extract of Radix chinensis, and for composition G, The bacteriostatic effect of doxycycline alone on Aeromonas hydrophila was lower than that of the composition that added the extract of Radix Glycyrrhizae or G. Only the composition of the extract can achieve the best bacteriostatic effect against Aeromonas hydrophila.

Example 4 The antibacterial effect of the composition containing the extract of Radix Glycyrrhizae and the extract of C. chinensis on Aeromonas Schubert strains, Aeromonas hydrophila and Aeromonas pipiens

After smashing and sieving the Scutellaria japonica powder, accurately weigh 600g of the powder and heat it at 80°C for 20 minutes; then divide the heated powder into three parts of the same quality of Scutellaria japonica powder. The blood vine powder was added to 20 times the amount of ethanol with a concentration of 50%, and extracted for 12 hours to obtain an extract A. The second part of the vine root powder was added to 20 times the amount of ethanol with a concentration of 60%. After leaching for 12 hours, the extraction solution B was obtained, and the third part of the powder was added to 20 times the amount of ethanol with a concentration of 90%, and the extraction was performed for 48 hours to obtain the extraction solution C; The solution A, the extract B and the extract C are mixed evenly and filtered, and the obtained filtrate is rotary steamed to obtain the Scutellaria chinensis extract;

After crushing and sieving Qianjinpu, get Qianjinpu powder, accurately weigh 1500g Qianjinpu powder and add it to 20 times the amount of ethanol with a concentration of 70%, extract for 2 hours, and reflux the obtained extract at 80 ° C for extraction , and then concentrated to obtain the extract of Qianjinpu;

Mix 48 g of the extract of Scutellaria officinalis and 90 g of the extract of Qianjinpu, and then add 20 mg of doxycycline to them and mix them evenly to obtain the composition.

Bacterial suspensions of Aeromonas Schubert, Aeromonas hydrophila and Aeromonas pipiens were added to different test tubes to make the bacterial concentration reach 103CFU/mL. The composition was added as a blank control, and no bacteria was added as a negative control, and each was repeated 3 times. Placed in a shaker at 28°C and shaken (180r/min) for 24h, and then taken out and compared with the control tube for observation.

Table 3 The antibacterial effect of the composition on different Aeromonas bacteria

In the above table, "+++" indicates extreme sensitivity, "++" indicates high sensitivity, "+" indicates moderate or low sensitivity, and "-" indicates insensitive (no antibacterial effect).

As can be seen from Table 3, the composition containing doxycycline, extract of Radix Glycyrrhizae and extract of Qianjinpu has significant effects on Aeromonas Schubert strains, Aeromonas hydrophila and Aeromonas pipiens. Bacteriostatic effect.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (3)

1. A composition for inhibiting aeromonas, comprising: the composition is prepared from the following raw materials in parts by weight:

0.008 to 0.028 portion of doxycycline,

32-50 parts of a suberect spatholobus stem extract,

85-100 parts of philippine flemingia root extract;

the preparation method of the caulis spatholobi extract comprises the following steps:

(1) pulverizing caulis Spatholobi, sieving to obtain caulis Spatholobi powder, and heating the powder at 60-90 deg.C for 5-30 min;

(2) then dividing the heated powder into three parts of caulis Spatholobi powder with the same mass, adding the first part of caulis Spatholobi powder into 10-30 times of 40-55% ethanol, leaching for 12-24 hours to obtain leaching liquor A, adding the second part of caulis Spatholobi powder into 10-30 times of 60-75% ethanol, leaching for 12-24 hours to obtain leaching liquor B, adding the third part of caulis Spatholobi powder into 10-30 times of 80-95% ethanol, leaching for 24-48 hours to obtain leaching liquor C;

(3) uniformly mixing the leaching liquor A, the leaching liquor B and the leaching liquor C, filtering, and rotatably steaming the obtained filtrate to obtain the leaching liquor;

the temperature of the heating step in the step (1) is 60-80 ℃, and the time is 15-30 minutes; adding the first part of caulis spatholobi powder in the step (2) into 50-55% ethanol, adding the second part of caulis spatholobi powder into 60-65% ethanol, and adding the third part of caulis spatholobi powder into 80-90% ethanol;

the philippine flemingia root extract is prepared by the following steps: pulverizing and sieving philippine flemingia root to obtain philippine flemingia root powder, adding the philippine flemingia root powder into 10-30 times of 60-70% ethanol, leaching for 1-2 hours, performing reflux extraction on the obtained leaching solution at 80-90 ℃, and concentrating to obtain a philippine flemingia root extract;

the aeromonas is schubert aeromonas strain, aeromonas hydrophila and aeromonas spot.

2. A process for preparing a composition for inhibiting the genus aeromonas according to claim 1, wherein: the method comprises the following steps: mixing caulis Spatholobi extract and radix Flemingiae Philippinensis extract, adding doxycycline, and mixing.

3. Use of an aeromonas-inhibiting composition of claim 1 in the manufacture of a medicament for inhibiting an aeromonas bacterium, wherein: the aeromonas is schubert aeromonas strain, aeromonas hydrophila and aeromonas spot.