Preparation method of sea-buckthorn branch and leaf water extract and application of sea-buckthorn branch and leaf water extract in inhibiting aquatic pathogenic bacteria

Abstract

The invention discloses a preparation method of a hippophae rhamnoides branch and leaf water extract and application of the hippophae rhamnoides branch and leaf water extract in inhibition of aquatic pathogenic bacteria. The preparation method comprises the following steps: drying and crushing fresh hippophae rhamnoides leaves or hippophae rhamnoides branches; adding water, and carrying out condensation reflux extraction; removing the solvent in the filtrate; and finally, grinding into powder, namely the sea-buckthorn branch and leaf water extract. The method not only makes full use of sea-buckthorn resources, but also has the advantages of safety, simplicity, convenience and low cost, and is easy to popularize on a large scale; the prepared hippophae rhamnoides branch and leaf water extract shows a good inhibition effect on vibrio aquatic pathogenic bacteria and particularly has an obvious inhibition effect on vibrio parahaemolyticus, the MIC of the hippophae rhamnoides branch water extract reaches 6.25 mg / mL, and the MIC of the hippophae rhamnoides leaf water extract reaches 1.56 mg / mL; the invention can be applied to preparation of aquatic pathogenic bacteria resistant drugs or feed additives, provides a powerful means for control and prevention of vibriosis of aquatic animals, and has important practical value in the aspect of guaranteeing the safety of aquaculture.

Description

Technical Field

[0001] This invention relates to a method for preparing and applying an extract of sea buckthorn branches and leaves, and more particularly to a method for preparing an aqueous extract of sea buckthorn branches and leaves and its application in inhibiting aquatic pathogens. Background Technology

[0002] In aquaculture, pathogenic infections are a major cause of disease and death in farmed animals, resulting in significant economic losses. Diseases caused by pathogens such as Vibrio are particularly common, especially Vibrio mimicry (Vibrio spp.). V. mimicus ), Vibrio parahaemolyticus ( V. parahaemolyticus ), Vibrio alginolyticus ( V. alginolyticus Aquatic pathogens, such as those causing intestinal infections and septicemia, can invade farmed animals through contaminated water and feed, severely hindering the healthy and sustainable development of aquaculture. The primary means of controlling aquatic pathogen infections is the use of antibiotics. However, long-term and excessive use of antibiotics not only easily leads to drug resistance in pathogens, gradually reducing the effectiveness of drug treatment, but also results in antibiotic residues in aquaculture water and aquatic products. These residues accumulate through the food chain, threatening the ecological environment and human health. Furthermore, the types of natural antibacterial agents targeting aquatic pathogens are limited, and some products suffer from unsatisfactory antibacterial effects and complex preparation processes. Therefore, the aquaculture industry urgently needs to find safer, more efficient, and environmentally friendly natural antibacterial substances.

[0003] Plant extracts, as natural products, have advantages such as wide availability, good biocompatibility, and low likelihood of inducing drug resistance, showing promising application prospects in the field of antibacterial agents. Sea buckthorn is a plant used for both food and medicine, rich in various bioactive components such as flavonoids, phenols, vitamins, and amino acids. It has significant effects in antioxidation, anti-inflammation, and enhancing immunity, and is widely studied and applied in food, medicine, and other fields. The most commonly used parts are sea buckthorn fruit and seeds. In addition, sea buckthorn leaves can be used to produce health teas, animal feed, and for extracting functional components. Patent application CN 120478705 A discloses a method for extracting and applying polyphenols from sea buckthorn leaves, using an enzymatic pretreatment combined with an ultrasonic-induced cell-wall breaking-step extraction system to improve polyphenol yield. However, because this method only extracts polyphenols, it does not fully utilize other valuable components in sea buckthorn leaves, and requires the use of enzymes and organic solvents, making the process complex and costly. Furthermore, research and utilization of other parts such as sea buckthorn branches are relatively scarce, in addition to fruits, seeds, and leaves. In particular, there are no reports of applications of sea buckthorn leaves or branches in combating aquatic pathogens. Summary of the Invention

[0004] Purpose of the invention: One purpose of the invention is to provide a method for preparing a water extract of sea buckthorn branches or a water extract of sea buckthorn leaves, and another purpose is to provide the application of the water extract in inhibiting aquatic pathogens.

[0005] Technical solution: The preparation method includes the following steps: (1) Dry fresh sea buckthorn leaves or branches at 50℃-60℃ and then pulverize them into powder; (2) The powder is mixed with water at a solid-liquid ratio of 1:6 to 1:8 (kg / L), and extracted by reflux to obtain filtrate; (3) Remove the solvent from the filtrate to obtain the crude extract; (4) Grind the crude extract into powder, which is the water extract of sea buckthorn branches and leaves.

[0006] In step (2), the reflux temperature is 100~105℃, preferably 100℃; the number of extractions is no less than 3 times, preferably 3 times; and the extraction time for each extraction is 2~2.5 h, preferably 2 h.

[0007] In step (3), the preferred method for removing the solvent is vacuum concentration, water bath or vacuum drying; the temperature of vacuum concentration is 55℃-60℃, preferably 60℃; the temperature of water bath or vacuum drying is 65℃-70℃, preferably 70℃; the preferred method for removing the solvent is: first, vacuum concentration at 60℃, then drying in a 70℃ water bath until viscous, and then vacuum drying at 70℃.

[0008] The application refers to the use of the water extract of sea buckthorn branches and leaves in inhibiting aquatic pathogens, or in the preparation of drugs or feed additives that combat aquatic pathogens. The aquatic pathogens are Vibrio mimicus, Vibrio parahaemolyticus, and Vibrio alginolyticus.

[0009] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The preparation method provided by this invention helps to make full use of sea buckthorn resources; compared with organic solvent extraction, this method avoids the use of hazardous chemicals and improves the safety of application; at the same time, this method has a simple process and is easy to operate, which greatly reduces production costs and has the advantage of being easy to promote on a large scale, thus promoting the development of related industries. 2. This method provides a novel and efficient natural antibacterial solution for aquaculture. The prepared water extracts of sea buckthorn branches and leaves showed good inhibitory effects on Vibrio mimicry, Vibrio parahaemolyticus, and Vibrio alginolyticus. Among them, the inhibitory effect on Vibrio parahaemolyticus was particularly significant, with the MIC of sea buckthorn branch water extract being 6.25 mg / mL and the MIC of sea buckthorn leaf water extract being 1.56 mg / mL. 3. This invention can be applied to inhibiting aquatic pathogens, including the preparation of anti-aquatic pathogen drugs or feed additives, providing a powerful means for the control and prevention of vibriosis in aquatic animals, and has important practical value in ensuring the safety of aquaculture. Attached Figure Description

[0010] Figure 1 The image shows the antibacterial effect of sea buckthorn branch water extract on five common aquatic pathogens. Figure 2 The image shows the antibacterial effect of sea buckthorn leaf water extract against five common aquatic pathogens. Figure 3 This diagram illustrates the minimum inhibitory concentrations of water extracts from sea buckthorn branches and leaves. Detailed Implementation

[0011] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0012] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0013] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0014] Example 1

[0015] This embodiment provides a method for preparing a water extract of sea buckthorn branches and leaves, the steps of which are as follows: (1) Place fresh sea buckthorn leaves or branches (collected in Huangyuan County, Qinghai Province) in an oven and dry them at 50°C until constant weight. Then, grind them into powder using a grinder and store them in a sealed container away from light for later use. (2) Accurately weigh the above sea buckthorn leaf powder, add pure water at solid-liquid ratios (kg / L) of 1:8 and 1:6 respectively, place in a round-bottom flask, and perform reflux extraction (100℃, repeated 3 times, 2 h each time) to obtain sea buckthorn leaf filtrate; sea buckthorn branch powder is treated in the same way to obtain sea buckthorn branch filtrate. (3) The above filtrate was transferred to a rotary evaporator and concentrated to a certain volume under reduced pressure at 60°C. The concentrate was then transferred to a beaker and heated in a 70°C water bath until it became viscous. The viscous material was then transferred to a 70°C vacuum drying oven and dried for 6-8 hours to obtain the crude extract of sea buckthorn leaves. The filtrate of sea buckthorn branches was treated in the same way to obtain the crude extract of sea buckthorn branches. (4) Use a fully automatic sample rapid grinder to grind the crude extract into a uniform powder to obtain the water extract of sea buckthorn leaves; the crude extract of sea buckthorn branches is treated in the same way to obtain the water extract of sea buckthorn branches; after dispensing, store in the dark and cold.

[0016] The yields of each group (yield = powder mass / water extract mass) are shown in Table 1. When the solid-liquid ratio (kg / L) is 1:8, the yield of sea buckthorn water extract is relatively good, at about 24%.

[0017] .

[0018] Example 2

[0019] This embodiment demonstrates the antibacterial effects of water extracts from sea buckthorn leaves and branches against common aquatic pathogens by detecting the minimum inhibitory concentration (MIC).

[0020] Preparation of the test solution: 100 mg of the water extract of sea buckthorn leaves obtained in Example 1 at a solid-liquid ratio of 1:8 was dissolved in 2 mL of sterile LB broth medium to obtain a final concentration of 50 mg / mL. The water extract of sea buckthorn branches was treated in the same way.

[0021] The experimental strains were five common aquatic pathogens: Enterobacter cloacae, Aeromonas verrucosa, Vibrio mimicus, Vibrio parahaemolyticus, and Vibrio alginolyticus, provided by Hohai University and Yangzhou University, respectively. Preparation: (1) Revitalization of the strain: Under aseptic conditions, the pathogens preserved in glycerol tubes were picked up with a sterile inoculation loop and inoculated onto LB + 2% NaCl medium plates by streak plating. The plates were then incubated at 30°C for 8-12 h. After single colonies grew, single colonies were selected and streak-plated again for purification. This process was repeated 2-3 times to obtain a stable pure culture. The plates were sealed with sealing film and stored in a 4°C refrigerator. (2) Preparation of bacterial suspension: Single colonies were picked from LB + 2% NaCl solid medium cultured for 8-12 h and inoculated into 50 mL of LB + 2% NaCl broth medium. The culture was shaken overnight at 30°C and 180 rpm to rapidly amplify the bacteria. Using a blank culture medium as a control, the bacterial suspension concentration was adjusted at 600 nm to achieve an absorbance of approximately 0.5, at which point the bacterial cell density was approximately 1 × 10⁻⁶. 8 CFU / mL. The bacterial culture was then diluted 1000-fold to obtain a density of 1×10⁻⁶. 5 Prepare and use fresh bacterial suspensions at CFU / mL.

[0022] The minimum inhibitory concentration (MIC) was determined using a two-fold serial dilution method: the test drug solution was prepared sequentially to concentrations of 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, and 0.12 mg / mL using the two-fold serial dilution method; 100 μL of each of the above different concentrations was added to the corresponding wells of a 96-well plate, followed by the addition of 100 μL of a 10% concentration to each well. 5The bacterial suspension was prepared at CFU / mL, making the total volume of each well 200 μL. To address the issue of the extract's dark color making the results difficult to observe, a control row was set up: 100 μL of the same concentration of the drug solution and 100 μL of the same volume of liquid culture medium (as a substitute for the bacterial suspension) were added to each well for comparison. After capping, the 96-well plates were incubated at 30℃ for 8–12 h. After incubation, the antibacterial effect was initially assessed by observing whether the bacterial suspension in each well was turbid. Then, 10 μL of MTT solution was added to each well for staining. After the addition was complete, the 96-well plates were incubated at 37℃ for 1–2 h, and the color change of the wells was observed visually: if the well was stained blue, it indicated bacterial growth; if it was not stained blue, it indicated no bacterial growth. The experimental results were recorded, and the lowest concentration at which no deep blue-purple formazan crystals appeared was recorded as the MIC of the water extracts of Hippophae rhamnoides branches and leaves.

[0023] The results are shown in Table 2. Figures 1-3 As shown, both the aqueous extracts of sea buckthorn branches and leaves exhibited good antibacterial activity against Vibrio aquatic pathogens, with the leaf extract showing superior antibacterial effect compared to the branch extract. The inhibitory effect against Vibrio parahaemolyticus was particularly significant, with the MICs for the branch and leaf extracts being 6.25 mg / mL and 1.56 mg / mL, respectively.

[0024] .

Claims

1. A method for preparing an aqueous extract of sea buckthorn branches and leaves, characterized in that, Includes the following steps: (1) Dry fresh sea buckthorn leaves or branches at 50℃-60℃ and then pulverize them into powder; (2) The powder is mixed with water at a solid-liquid ratio of 1:6 to 1:8 (kg / L), and extracted by reflux to obtain filtrate; (3) Remove the solvent from the filtrate to obtain the crude extract; (4) Grind the crude extract into powder, which is the water extract of sea buckthorn branches and leaves.

2. The preparation method according to claim 1, characterized in that, In step (2), the temperature of the condensation reflux is 100~105℃, and the number of extractions is no less than 3.

3. The preparation method according to claim 2, characterized in that, Each extraction takes 2 to 2.5 hours.

4. The preparation method according to claim 1, characterized in that, In step (3), the solvent removal methods include vacuum concentration, water bath or vacuum drying.

5. The preparation method according to claim 4, characterized in that, The temperature for vacuum concentration is 55℃-60℃.

6. The preparation method according to claim 4, characterized in that, The temperature of the water bath is 65℃-70℃.

7. The preparation method according to claim 4, characterized in that, The vacuum drying temperature is 65℃-70℃.

8. The application of the water extract of sea buckthorn branches and leaves according to claim 1 in inhibiting aquatic pathogens.

9. The application according to claim 8, characterized in that, The aquatic pathogens mentioned are Vibrio mimicus, Vibrio parahaemolyticus, and Vibrio alginolyticus.

10. The use of the water extract of sea buckthorn branches and leaves according to claim 1 in the preparation of drugs or feed additives for combating aquatic pathogens.

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