Application of water chestnut hull extract in preparation of medicines for inhibiting marine pathogenic bacteria

Abstract

The invention relates to the technical field of Chinese herbal medicine screening and extraction and separation, in particular to application of a water chestnut hull extract, namely application of the water chestnut hull extract in preparation of drugs for inhibiting marine pathogenic bacteria. The marine pathogenic bacteria are Vibrio harveyi or Vibrio parahaemolyticus, and the marine pathogenic bacteria are the Vibrio harveyi or the Vibrio parahaemolyticus. The water chestnut hull extract is obtained by taking water chestnut hulls as raw materials and adopting water extraction or alcohol extraction. Compared with the prior art, the preparation method has the following beneficial effects that the water chestnut hull extract is used for preparing the medicine for inhibiting the marine pathogenic bacteria, the water chestnut hull alcohol extract has a relatively good inhibition effect on vibrio harveyi and vibrio parahaemolyticus, and the MIC values are 31.25 mg / mL and 15.63 mg / mL respectively.

Description

Technical Field

[0001] The present invention relates to the technical field of screening and extraction and separation of Chinese herbal medicines, and particularly relates to the use of water chestnut shell extract in the preparation of drugs for inhibiting marine pathogenic bacteria. Background Art

[0002] As the world's largest aquaculture country, China's annual aquaculture output accounts for about 70% of the world's total aquaculture output. However, with the increasing pollution of the aquaculture environment, the outbreak of aquatic animal diseases, the increase in the types of pathogenic microorganisms, and the acceleration of the transmission speed have occurred. Currently, bacterial diseases have a very high incidence in aquaculture, especially in intensive aquaculture systems, seriously hindering the aquaculture and development of marine fish, shrimps, and shellfishes. Diseases caused by pathogenic Vibrio have a wide epidemic area and a high incidence rate, causing huge harm to the aquaculture industry. Marine pathogenic bacteria mainly include Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum, Vibrio alginolyticus, etc.

[0003] Currently, for marine pathogenic bacteria, chemical drug prevention and control methods are mostly adopted, mainly including penicillin, norfloxacin, sulfamethoxazole, etc. However, long-term single use of a drug will produce resistance and drug-resistant strains will appear. Chinese herbal medicines have the advantages of rich components, not being prone to drug resistance, being green and environmentally friendly, and having diverse effects, and have unique effects and good effects in the prevention and control of various aquatic diseases such as viral and bacterial diseases.

[0004] Currently, there have been studies on screening Chinese herbal medicines to inhibit marine pathogenic bacteria, but there is currently a lack of reports on the application of the Chinese herbal medicines or food processing waste of the present invention in inhibiting marine pathogenic bacteria. Therefore, screening Chinese herbal medicines and finding effective Chinese herbal medicines with good inhibition of marine pathogenic bacteria is of great significance.

[0005] Water chestnut shell is the outer seed coat of the water chestnut (Trapa bispinosa Roxb.) of the genus Trapa in the family Trapaceae. The pulp of water chestnut has high nutritional value, but in the process of food processing, water chestnut shell is discarded in large quantities as a by-product. Water chestnut shell is hard in texture, with two horizontally spreading shoulder angles, showing a curved ox-horn shape, without or with barbs, the tip is bent downward, the width between the two angles is 7-8 cm, showing a curved ox-horn shape, the fruit height is 2.5-3.6 cm, the young skin of the fruit surface is purple-red, and when it is fully mature, it is purple-black, slightly covered with extremely short hairs. As an annual aquatic herbaceous plant, it is produced throughout the country, with the most distribution in the subtropical regions of the Yangtze River Basin. Summary of the Invention

[0006] The purpose of the present invention is to provide the use of water chestnut shell extract in the preparation of drugs for inhibiting marine pathogenic bacteria in view of the deficiencies of the prior art.

[0007] The technical problems to be solved by the present invention are realized through the following technical solutions:

[0008] The inventors of the present invention have first discovered a new use of the water chestnut shell extract, and the said use is the use of the water chestnut shell extract in the preparation of drugs for inhibiting marine pathogenic bacteria; the said marine pathogenic bacteria are Vibrio harveyi or Vibrio Parahaemolyticus.

[0009] Preferably, the said water chestnut shell extract is a water chestnut shell extract obtained by using water chestnut shells as raw materials and adopting water extraction or alcohol extraction.

[0010] Preferably, the preparation method of the said water chestnut shell extract is as follows: Weigh the water chestnut shell powder, add the solvent water or ethanol, ultrasonically extract at 30-60°C for 10-90 minutes, then reflux extract at 30-90°C for 30-120 minutes, filter by suction, extract 1-4 times in the same way, collect the filtrate, and concentrate to obtain.

[0011] Preferably, the said water chestnut shell extract is an extract mainly composed of phenolic acid compounds extracted from water chestnut shells.

[0012] More preferably, the extraction method of the said water chestnut shell extract is as follows:

[0013] (1) Extraction: Weigh the water chestnut shell powder, add ethanol, ultrasonically extract at 50°C for 30 minutes, then reflux extract at 50°C for 30 minutes, filter by suction, extract 2 times in the same way, collect the filtrate, and concentrate to make the final concentration of the crude drug 1 g / mL;

[0014] (2) Separation of antibacterial active ingredients: Separate the ethanol extract of water chestnut shells. After the ethanol extract of water chestnut shells is rotary evaporated into an extract, add distilled water to make a turbid solution, and extract with equal volumes of petroleum ether, ethyl acetate, and n-butanol respectively until the solution is clear and colorless. The extraction time for each time is 30 minutes. After the extraction is completed, rotary dry the solvent to obtain a dry powder. For the dry powders of different extracts, prepare a sample solution of 10 mg / mL, and use the Oxford cup method to determine the antibacterial activity of the sample solution;

[0015] (3) Chemical qualitative detection of antibacterial active ingredients: Use chemical qualitative methods to detect the four components of the petroleum ether group, ethyl acetate group, n-butanol group, and distilled water group, and use hydrochloric acid + magnesium powder, bromocresol green solution, and ferric chloride-potassium ferricyanide solution for chemical qualitative detection; determine that the ethyl acetate group is phenolic acid compounds; determine that phenolic acid compounds are the effective components for inhibiting marine pathogenic bacteria.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention discovers for the first time that the extract of water chestnut shell can be used to prepare a drug for inhibiting marine pathogenic bacteria. In particular, the ethanol extract of water chestnut shell has good inhibitory effects on Vibrio harveyi and Vibrio parahaemolyticus, and the MIC values are 31.25 mg / mL and 15.63 mg / mL respectively. Detailed implementation manners

[0017] The following provides a detailed description of the specific implementation manners of the present invention in conjunction with the embodiments.

[0018] The sources of the materials used in the present invention are as follows: Cortex Magnoliae Officinalis Praeparata, water chestnut shell, Rheum officinale, Concha Ostreae, Artemisia argyi Levl. et Vant., Paeonia lactiflora Pall., Fructus Aurantii Immaturus, Concha Arcae, Ardisia japonica (Thunb.) Blume, Radix Aconiti Lateralis Praeparata, Calyx Kaki, Phyllanthus niruri Linn., Bombax ceiba Linn., Fructus Toosendan, all are purchased from Bozhou Mingxu Pharmaceutical Sales Co., Ltd. Vibrio harveyi (V. harveyi 1.8690) and Vibrio parahaemolyticus (V. parahaemolyticus 1.1997) are both purchased from the China General Microbiological Culture Collection Center.

[0019] Example 1

[0020] Screen traditional Chinese medicines with the effect of inhibiting marine pathogenic bacteria, and determine that water chestnut shell is the traditional Chinese medicine with the best activity in inhibiting marine pathogenic bacteria.

[0021] 1. Screening of traditional Chinese medicines for inhibiting marine pathogenic bacteria

[0022] Initially select the water extracts and ethanol extracts of Cortex Magnoliae Officinalis Praeparata, water chestnut shell, Rheum officinale, Concha Ostreae, Artemisia argyi Levl. et Vant., Paeonia lactiflora Pall., Fructus Aurantii Immaturus, Concha Arcae, Ardisia japonica (Thunb.) Blume, Radix Aconiti Lateralis Praeparata, Calyx Kaki, Phyllanthus niruri Linn., Bombax ceiba Linn., Fructus Toosendan, a total of 14 traditional Chinese medicines. The preparation methods of the water extracts and ethanol extracts of each traditional Chinese medicine are as follows: Weigh the traditional Chinese medicine powder, add the solvent (water or ethanol), ultrasonically extract at 50 °C for 30 min, then reflux extract at 50 °C for 30 min, filter by suction, extract 2 times in the same way, collect the filtrate, and concentrate to make the final concentration of the crude drug 1 g / mL.

[0023] Select Vibrio harveyi and Vibrio parahaemolyticus as the target strains. Using the Oxford cup method, in a sterile environment, pour about 20 mL of sterilized MH broth medium into the petri dish. After solidification, add 200 μL of the bacterial suspension, spread evenly with a spreading rod, and let stand for 10 min. Place the Oxford cups vertically and evenly on the medium, add 200 μL of the extract sample into the cups, conduct 3 parallel experiments for each sample, transfer to a 37 °C constant temperature incubator for 18 h, and then measure the diameter of the inhibition zone with an electronic digital caliper. The result is the average value of the three measurement results. An inhibition zone diameter greater than 20 mm is extremely sensitive, 15 - 19 mm is highly sensitive, 10 - 15 mm is moderately sensitive, and less than 10 mm is lowly sensitive (inactive).

[0024] Through the above in vitro antibacterial experiments, Chinese herbal medicines with strong inhibitory effects on Vibrio harveyi and Vibrio parahaemolyticus were selected as the subjects for further experiments.

[0025] Table 1 Antibacterial effects of 14 Chinese herbal medicine extracts on two Vibrio species

[0026]

[0027]

[0028] Note: "+++" indicates extremely sensitive, "++" indicates highly sensitive, "+" indicates moderately sensitive, and "-" indicates low sensitivity (inactive).

[0029] The experimental results are shown in Table 1. The diameters of the antibacterial zones of ethanol extracts of Magnolia officinalis Rehd. var. biloba (Rehd.) Cheng et Law, Asarum sieboldii Miq., Rheum palmatum L., and water extracts of Trapa bispinosa Roxb. shells, Asarum sieboldii Miq. against one or both of Vibrio parahaemolyticus and Vibrio harveyi reached the highly sensitive level. The diameters of the antibacterial zones of ethanol extracts or water extracts of Magnolia officinalis Rehd. var. biloba (Rehd.) Cheng et Law, Rheum palmatum L., Blumea balsamifera (L.) DC., Paeonia lactiflora Pall., Phyllanthus niruri Linn., Ardisia japonica (Thunb.) Blume, Calyx kaki Thunb., Bombax malabaricum DC., Melia azedarach L., and Fructus Aurantii Immaturus against one or both of Vibrio parahaemolyticus and Vibrio harveyi reached the moderately sensitive level. The ethanol extract of Trapa bispinosa Roxb. shells had the strongest inhibitory activity against the two Vibrio species, with the largest antibacterial zone diameters, which were (23.30 ± 0.31) mm and (21.92 ± 0.20) mm, respectively.

[0030] According to the above experimental results, the ethanol extract of Trapa bispinosa Roxb. shells with the best inhibitory activity against Vibrio harveyi and Vibrio parahaemolyticus was selected as the subject for further experiments to determine its minimum inhibitory concentration (MIC).

[0031] 2. Determination of the MIC of the ethanol extract of Trapa bispinosa Roxb. shells against two Vibrio species

[0032] The MIC of the ethanol extract of Trapa bispinosa Roxb. shells against two Vibrio species was determined by the two-fold dilution method, that is, the ethanol extract of Trapa bispinosa Roxb. shells at a concentration of 1 g / mL was diluted to the crude drug concentrations of 500 mg / mL, 250 mg / mL, 125 mg / mL, 62.5 mg / mL, 31.25 mg / mL, 15.63 mg / mL, and 7.82 mg / mL. After culturing at 30 °C with constant shaking at 200 r / min for 24 h, the results were observed, and the concentration of the drug solution with the highest dilution without bacterial growth was taken as the MIC value.

[0033] Table 2 Experimental results of the minimum inhibitory concentration of the ethanol extract of Trapa bispinosa Roxb. shells against two Vibrio species

[0034]

[0035]

[0036] Note: "-" indicates inactive.

[0037] The experimental results are shown in Table 2. The ethanol extract of water chestnut shell has good inhibitory effects on Vibrio harveyi and Vibrio parahaemolyticus, and the MIC values are 31.25 mg / mL and 15.63 mg / mL, respectively.

[0038] Example 2

[0039] Extraction, separation, detection and analysis of the active ingredients in water chestnut shell against marine pathogenic bacteria.

[0040] 1. Extraction of antibacterial active ingredients

[0041] Weigh the water chestnut shell powder, add ethanol, ultrasonically extract it at 50 °C for 30 min, then reflux extract it at 50 °C for 30 min, filter by suction, extract twice in the same way, collect the filtrate, and concentrate it to make the final concentration of the crude drug 1 g / mL.

[0042] 2. Separation of antibacterial active ingredients

[0043] Separate the ethanol extract of water chestnut shell. After the ethanol extract of water chestnut shell is rotary evaporated into an extract, add distilled water to make it into a turbid solution, and extract it with equal volume of petroleum ether, ethyl acetate, and n-butanol respectively until the solution becomes clear and colorless. The extraction time for each time is 30 min. After the extraction is completed, rotary evaporate the solvent to obtain a dry powder. Prepare the dry powders of different extracts into a sample solution of 10 mg / mL. Select Vibrio parahaemolyticus as the target strain. Using the Oxford cup method, in a sterile environment, pour about 20 mL of sterilized MH broth medium into the petri dish. After solidification, add 200 μL of the bacterial suspension, spread it evenly with a spreading rod, and let it stand for 10 min. Place the Oxford cups vertically and evenly on the medium, add 200 μL of the sample solution into each cup respectively, conduct 3 parallel experiments for each sample, transfer it to a 37 °C constant temperature incubator for 18 h, and then use an electronic digital caliper to measure the diameter of the inhibition zone. The result is the average value of the three measurement results. An inhibition zone diameter greater than 20 mm is extremely sensitive, 15 - 19 mm is highly sensitive, 10 - 15 mm is moderately sensitive, and less than 10 mm is low sensitive (inactive).

[0044] Table 3 Results of the inhibition of Vibrio parahaemolyticus by each component of water chestnut shell

[0045] Test strains Petroleum ether group Ethyl acetate group n-Butanol group Distilled water group Vibrio parahaemolyticus 8.21 20.10(+++) 12.11(+) 12.08(+)

[0046] Note: "+++" indicates extremely sensitive, "++" indicates highly sensitive, "+" indicates moderately sensitive, and "-" indicates low sensitive (inactive).

[0047] Through the separation of different polarity segments, four components are obtained. Conduct antibacterial experiments on the four components. The experimental results are shown in Table 3. The petroleum ether group has no inhibitory activity against Vibrio parahaemolyticus, and both the n-butanol group and the distilled water group have inhibitory effects on Vibrio parahaemolyticus. The ethyl acetate group has good inhibitory effects on Vibrio parahaemolyticus.

[0048] 3. Chemical Qualitative Detection of Bacteriostatic Active Ingredients

[0049] The ethyl acetate group was detected by chemical qualitative methods, and chemical qualitative detections were carried out using hydrochloric acid + magnesium powder, bromocresol green solution and ferric chloride - potassium ferricyanide solution.

[0050] Table 4 Detection of Active Ingredients in Water Chestnut Shells against Vibrio parahaemolyticus

[0051] Isolated fraction Hydrochloric acid + magnesium powder Bromocresol green solution Ferric chloride - potassium ferricyanide solution Ethyl acetate group No reaction Yellow Blue

[0052] The experimental results are shown in Table 4. The ethyl acetate group showed a positive reaction with bromocresol green solution, indicating that its main component is carboxylic acid compounds; the ethyl acetate group also showed a positive reaction with ferric chloride - potassium ferricyanide solution, indicating that its main component is phenolic acid compounds.

[0053] Through the above separation and chemical qualitative detection, it was preliminarily determined that the effective ingredient in water chestnut shells inhibiting marine pathogenic bacteria is phenolic acid compounds.

[0054] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the method of the present invention, several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. The use of water chestnut shell extract, characterized in that: The use is the use of the water chestnut shell extract in the preparation of drugs for inhibiting marine pathogenic bacteria; the marine pathogenic bacteria are Vibrio harveyi or Vibrio Parahaemolyticus.

2. The use of the water chestnut shell extract according to claim 1, characterized in that The water chestnut shell extract is obtained by taking water chestnut shell as raw material and adopting water extraction or alcohol extraction.

3. The use of the water chestnut shell extract according to claim 1, characterized in that The preparation method of the water chestnut shell extract is as follows: weigh water chestnut shell powder, add solvent water or ethanol, ultrasonically extract at 30-60°C for 10-90min, then reflux extract at 30-90°C for 30-120min, filter, extract 1-4 times in the same way, collect the filtrate, and concentrate to obtain the extract.

4. The use of the water chestnut shell extract according to claim 1, characterized in that The water chestnut shell extract is an extract obtained from water chestnut shells, the main component of which is phenolic acid compounds.

5. The use of the water chestnut shell extract according to claim 1, characterized in that The method for extracting the water chestnut shell extract and separating the antibacterial active components is as follows: (1) Extraction: Weigh water chestnut shell powder, add ethanol, extract by ultrasonic at 50°C for 30 min, then extract by reflux at 50°C for 30 min, filter, extract twice in the same way, collect the filtrate, and concentrate it to make the final concentration of the crude drug 1 g / mL; (2) Isolation of antibacterial active ingredients: The alcohol extract of water chestnut shell is separated, and the alcohol extract of water chestnut shell is rotary evaporated to obtain an extract, and distilled water is added to prepare a turbid liquid, which is extracted with an equal volume of ethyl acetate until the solution is clear and colorless, and the extraction time is 30 min. After the extraction is completed, the solvent is rotary dried to obtain a dry powder.

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