An antibacterial agent containing an extract of acacia leaf for inhibiting vibrio alginolyticus and application thereof

Abstract

The application provides an antibacterial agent containing an extract of Aquilaria sinensis leaves for inhibiting Vibrio alginolyticus and an application thereof, and relates to the field of plant extracts. The antibacterial agent is the extract of Aquilaria sinensis leaves; the extract of Aquilaria sinensis leaves is prepared by crushing Aquilaria sinensis leaves with water, the ratio of the Aquilaria sinensis leaves to water is 1g:2-4mL, and the extract of Aquilaria sinensis leaves is used for inhibiting Vibrio alginolyticus. The preparation method of the extract of Aquilaria sinensis leaves comprises the following steps: taking Aquilaria sinensis leaves, adding water, crushing into a homogenate, filtering impurities, centrifuging, collecting supernatant, sterilizing, and obtaining the extract of Aquilaria sinensis leaves. The minimum inhibitory concentration of the extract of Aquilaria sinensis leaves prepared in the application for Vibrio alginolyticus is 15.63mg / ml. The extract of Aquilaria sinensis leaves prepared in the application can better inhibit Vibrio alginolyticus, is used for preparing the antibacterial agent for inhibiting Vibrio alginolyticus, avoids the generation of the risk of drug resistance genes, has no risk of organic solvent residue, and has many advantages such as high efficiency, reduced risk and reduced cost.

Description

An antibacterial agent containing agarwood leaf extract that inhibits Vibrio alginolyticus and its application. Technical Field

[0001] This invention relates to the field of plant extracts, and in particular to an antibacterial agent containing agarwood leaf extract that inhibits Vibrio alginolyticus and its application. Background Technology

[0002] Currently, antimicrobial treatment for Vibrio alginolyticus mainly relies on antibiotics, but this method carries the risk of generating drug-resistant genes, and antibiotics are expensive.

[0003] Agarwood (Latin name: *Aquilaria sinensis* (Lour.) Spreng.), also known as agarwood tree, is a tropical and subtropical evergreen tree belonging to the Thymelaeaceae family. Extraction from the leaves of the agarwood tree (referred to as agarwood leaves) traditionally utilizes a time-consuming reflux extraction method. Prolonged heating can cause denaturation of some of the active ingredients in the extract, leading to its loss. Furthermore, the use of chemical solvents such as ethanol for extraction significantly increases production costs. Summary of the Invention

[0004] In view of this, the present invention proposes an antibacterial agent containing agarwood leaf extract that inhibits Vibrio alginolyticus and its application.

[0005] The technical solution of this invention is implemented as follows:

[0006] An antibacterial agent, wherein the antibacterial agent is an extract of agarwood leaves; the agarwood leaf extract is obtained by crushing and extracting agarwood leaves (i.e., leaves of the agarwood tree Aquilaria sinensis (Lour.) Spreng.) with water, wherein the ratio of agarwood leaves to water is 1g:2-4mL, preferably 1g:4mL. The agarwood leaf extract is used to inhibit Vibrio alginolyticus. The agarwood leaf extract prepared by this invention can effectively inhibit Vibrio alginolyticus, and when used to produce an antibacterial agent against Vibrio alginolyticus, it avoids the risk of developing drug-resistant genes and has no risk of organic solvent residue, combining many advantages such as high efficiency, reduced risk, and reduced cost.

[0007] Furthermore, the preparation method of the agarwood leaf extract includes the following steps: taking agarwood leaves, adding water, crushing into a homogenate, filtering to remove impurities, centrifuging to separate, collecting the supernatant, sterilizing, and obtaining the agarwood leaf extract.

[0008] Furthermore, the preparation method of the agarwood leaf extract includes the following specific steps:

[0009] S1. Add fresh agarwood leaves to water and crush them into a homogenous slurry using a crusher to obtain agarwood leaf slurry.

[0010] S2. Filter the agarwood leaf pulp to remove impurities, and obtain agarwood leaf coarse filtrate;

[0011] S3. Centrifuge the coarse filtrate to separate it and collect the supernatant from the agarwood leaves;

[0012] S4. Filter the supernatant to obtain agarwood leaf extract;

[0013] S5. The agarwood leaf extract is sterilized by high temperature and high pressure to obtain agarwood leaf extract.

[0014] Further, in step S1, the crushing time using a crusher is 3-5 minutes.

[0015] Further, in step S2, the filtration is performed using gauze.

[0016] Further, in step S3, the centrifugation speed is 1700-2300 rpm and the centrifugation time is 8-12 minutes. Preferably, the centrifugation speed is 2000 rpm and the centrifugation time is 10 minutes.

[0017] Further, in step S4, the filtration is performed using filter paper.

[0018] Further, in step S5, the sterilization temperature is 120-122℃ and the sterilization pressure is 0.21-0.23MPa. Preferably, the sterilization temperature is 121℃ and the sterilization pressure is 0.22MPa.

[0019] The agarwood leaf extract described in any one of the present invention is used in the preparation of an antibacterial agent that inhibits Vibrio alginolyticus.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] (1) Further research conducted in this invention has revealed that the agarwood leaf extract prepared by crushing and extracting agarwood leaves can effectively inhibit Vibrio alginolyticus. The minimum inhibitory concentration of the agarwood leaf extract of this invention against Vibrio alginolyticus is 15.63 mg / ml.

[0022] (2) Compared with traditional antibiotics, the present invention uses agarwood leaf (natural plant leaf) extract to prepare antibacterial agent, which avoids the risk of drug resistance genes and achieves low-cost treatment.

[0023] (3) Compared with the reflux extraction method, the present invention uses the crushing extraction method to extract agarwood leaves for the production of antibacterial agents that inhibit Vibrio alginolyticus. This method is both fast and efficient.

[0024] (4) Compared with organic solvent extraction methods, the preparation method proposed in this invention has a simple process, is easy to operate, has obvious effects, high yield, significantly reduced cost, and is easy to promote on a large scale.

[0025] (5) The agarwood leaf extract of the present invention is an antibacterial agent for inhibiting Vibrio alginolyticus. It not only has no risk of organic solvent residue, but its preparation method is simple, easy to operate, and conducive to promotion and application. Attached Figure Description

[0026] Figure 1 shows the inhibition zones of agarwood leaf extract and imipenem against Vibrio alginolyticus in Example 1 of this invention. The inhibition zone on the left is the inhibition zone of the positive control (imipenem), and the inhibition zone on the right is the inhibition zone of agarwood leaf extract. Detailed Implementation

[0027] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods;

[0028] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0029] The ambient temperature range of this invention is 20-30℃.

[0030] Example 1

[0031] Raw materials for agarwood leaf extract: 20 grams of fresh agarwood leaves and 80 ml of purified water.

[0032] Preparation of agarwood leaf extract:

[0033] (1) Place fresh, disease-free agarwood leaves into a crusher, add purified water, grind at room temperature for 3 minutes to break them into a homogenous slurry, and obtain agarwood leaf water extract.

[0034] (2) The water extract of crushed agarwood leaves was filtered with gauze to remove coarse particles and obtain coarse filtrate of agarwood leaves.

[0035] (3) Transfer the coarse filtrate of agarwood leaves to a centrifuge tube and centrifuge at 2000 rpm for 10 minutes to separate the solid and liquid.

[0036] (4) Filter the supernatant after centrifugation with filter paper to remove residual impurities and obtain a clear agarwood leaf extract.

[0037] (5) The agarwood leaf extract was placed into a sterile container and sterilized under high temperature and high pressure for 30 minutes. The sterilization pressure was 0.22 MPa and the sterilization temperature was 121℃ to obtain the agarwood leaf extract, which is the agarwood leaf antibacterial solution.

[0038] Experimental Example 1

[0039] 1. Experimental method: The isolated Vibrio alginolyticus was picked and a single colony was prepared into a 0.5 McFarland suspension. The suspension was then inoculated into nutrient agar medium (spread plate method). After inoculation, two Oxford cups were placed in the petri dish. One cup was filled with 200 μL of 0.25 g / mL agarwood leaf extract, and the other cup was filled with 200 μL of imipenem. After standing for 3 min, the cups were placed in an incubator for incubation.

[0040] Test subject: Agarwood leaf extract (Example 1).

[0041] Positive control: Imipenem (antibiotic)

[0042] 2. Experimental Results

[0043] (1) Antibacterial activity of agarwood leaf extract against Vibrio alginolyticus

[0044] Table 1. Antibacterial activity against Vibrio alginolyticus

[0045]

[0046] Note: Determination of antibacterial effect: A diameter of ≥20mm indicates strong antibacterial effect, indicated by "+++"; 15mm~20mm indicates moderate antibacterial effect, indicated by "++"; 10mm~15mm indicates weak antibacterial effect, indicated by "+"; <10mm indicates "—".

[0047] The results showed that the agarwood leaf extract prepared in this invention has a strong antibacterial effect against Vibrio alginolyticus.

[0048] (2) Minimum inhibitory concentration

[0049] The concentration of the agarwood leaf extract in Example 1 was 250 mg / ml (based on the weight of the raw agarwood leaves). The minimum inhibitory concentration (MIC) of the agarwood leaf extract (Example 1) against *Vibrio alginolyticus* was determined using the two-fold dilution method in test tubes. Ten sterile capped test tubes were used and numbered. 1.0 ml of sterile broth was added to each tube. Then, 1.0 ml of sterile agarwood leaf extract was added to the first tube, mixed well, and 1.0 ml was transferred to the second tube. This serial dilution was continued until the eighth tube, at which point 1.0 ml was discarded. 1.0 ml of agarwood leaf extract was added to the ninth tube, mixed well, and 1.0 ml was discarded. This tube, without bacteria, served as a negative control. The tenth tube, without any added bacteria, served as a positive control. 0.5 ml of the test bacterial solution was added to each of the eight tubes and the positive control tube, and mixed well. 0.5 ml of sterile physiological saline was added to the ninth tube. All tubes were incubated in a constant temperature incubator for 24 hours, and the culture results were observed. Under the premise that bacterial growth in the positive control tube is turbid and bacterial growth in the negative control tube is clear, the turbidity of other test tubes is observed. If the test tube is turbid, it indicates bacterial growth; if the test tube is clear, it indicates no bacterial growth. The lowest concentration of drug solution with no bacterial growth is the minimum inhibitory concentration (MIC) of that drug against that bacterial strain. The results are shown in Table 2 below.

[0050] Table 2 Minimum Inhibitory Concentration

[0051]

[0052] Note: "+" indicates that bacterial growth is visible at this concentration; "-" indicates that no bacterial growth is visible at this concentration.

[0053] The results showed that the agarwood leaf extract of the present invention exhibited good inhibitory effects against Vibrio alginolyticus when the concentration was 15.63-250 mg / ml. The minimum inhibitory concentration of the agarwood leaf extract against Vibrio alginolyticus was 15.63 mg / ml.

[0054] Example 2

[0055] Raw materials for agarwood leaf extract: 20 grams of fresh agarwood leaves and 40 ml of purified water.

[0056] Preparation of agarwood leaf extract:

[0057] (1) Place fresh, disease-free agarwood leaves into a crusher, add purified water, grind at room temperature for 3 minutes to break them into a homogenous slurry, and obtain agarwood leaf water extract.

[0058] (2) The water extract of crushed agarwood leaves was filtered with gauze to remove coarse particles and obtain coarse filtrate of agarwood leaves.

[0059] (3) Transfer the coarse filtrate of agarwood leaves to a centrifuge tube and centrifuge at 2000 rpm for 10 minutes to separate the solid and liquid.

[0060] (4) Filter the supernatant after centrifugation with filter paper to remove residual impurities and obtain a clear agarwood leaf extract.

[0061] (5) The agarwood leaf extract was placed into a sterile container and sterilized under high temperature and high pressure for 30 minutes. The sterilization pressure was 0.22 MPa and the sterilization temperature was 121℃ to obtain the agarwood leaf extract, which is the agarwood leaf antibacterial solution.

[0062] Example 3

[0063] Raw materials for agarwood leaf extract: 20 grams of fresh agarwood leaves and 60 ml of purified water.

[0064] Preparation of agarwood leaf extract:

[0065] (1) Place fresh, disease-free agarwood leaves into a crusher, add purified water, grind at room temperature for 3 minutes to break them into a homogenous slurry, and obtain agarwood leaf water extract.

[0066] (2) The water extract of crushed agarwood leaves was filtered with gauze to remove coarse particles and obtain coarse filtrate of agarwood leaves.

[0067] (3) Transfer the coarse filtrate of agarwood leaves to a centrifuge tube and centrifuge at 2000 rpm for 10 minutes to separate the solid and liquid.

[0068] (4) Filter the supernatant after centrifugation with filter paper to remove residual impurities and obtain a clear agarwood leaf extract.

[0069] (5) The agarwood leaf extract was placed into a sterile container and sterilized under high temperature and high pressure for 30 minutes. The sterilization pressure was 0.22 MPa and the sterilization temperature was 121℃ to obtain the agarwood leaf extract, which is the agarwood leaf antibacterial solution.

[0070] Example 4

[0071] Raw materials for agarwood leaf extract: 20 grams of fresh agarwood leaves and 80 ml of purified water.

[0072] Preparation of agarwood leaf extract:

[0073] (1) Place fresh, disease-free agarwood leaves into a crusher, add purified water, grind at room temperature for 4 minutes to break them into a homogenous slurry, and obtain agarwood leaf water extract.

[0074] (2) The water extract of crushed agarwood leaves was filtered with gauze to remove coarse particles and obtain coarse filtrate of agarwood leaves.

[0075] (3) Transfer the coarse filtrate of agarwood leaves to a centrifuge tube and centrifuge at 1700 rpm for 12 minutes to separate the solid and liquid.

[0076] (4) Filter the supernatant after centrifugation with filter paper to remove residual impurities and obtain a clear agarwood leaf extract.

[0077] (5) The agarwood leaf extract was placed into a sterile container and sterilized under high temperature and high pressure for 30 minutes. The sterilization pressure was 0.22 MPa and the sterilization temperature was 121℃ to obtain the agarwood leaf extract, which is the agarwood leaf antibacterial solution.

[0078] Example 5

[0079] Raw materials for agarwood leaf extract: 20 grams of fresh agarwood leaves and 80 ml of purified water.

[0080] Preparation of agarwood leaf extract:

[0081] (1) Place fresh, disease-free agarwood leaves into a crusher, add purified water, grind at room temperature for 5 minutes to break them into a homogenous slurry, and obtain agarwood leaf water extract.

[0082] (2) The water extract of crushed agarwood leaves was filtered with gauze to remove coarse particles and obtain coarse filtrate of agarwood leaves.

[0083] (3) Transfer the coarse filtrate of agarwood leaves to a centrifuge tube and centrifuge at 2300 rpm for 8 minutes to separate the solid and liquid.

[0084] (4) Filter the supernatant after centrifugation with filter paper to remove residual impurities and obtain a clear agarwood leaf extract.

[0085] (5) The agarwood leaf extract was placed into a sterile container and sterilized under high temperature and high pressure for 30 minutes. The sterilization pressure was 0.22 MPa and the sterilization temperature was 121℃ to obtain the agarwood leaf extract, which is the agarwood leaf antibacterial solution.

[0086] The agarwood leaf extracts prepared in Examples 2-5 have a strong inhibitory effect on Vibrio alginolyticus.

[0087] Comparative Example 1

[0088] Following the method in Experimental Example 1, the antibacterial activity of agarwood leaf extract (Example 1) against other bacterial strains was studied, and the results are shown in Table 3 below.

[0089] Table 3 Antibacterial activity against different strains

[0090]

[0091] Note: Determination of antibacterial effect: A diameter of ≥20mm indicates strong antibacterial effect, indicated by "+++"; 15mm~20mm indicates moderate antibacterial effect, indicated by "++"; 10mm~15mm indicates weak antibacterial effect, indicated by "+"; <10mm indicates "—".

[0092] Studies have found that the agarwood leaf extract prepared in this invention has no inhibitory effect on Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis.

[0093] Comparative Example 2

[0094] A method for preparing an extract of agarwood stems and branches, using agarwood (Aquilaria sinensis (Lour.) Spreng.) stems and branches as raw materials, is employed by water decoction. 9.5g of agarwood stems and branches are weighed into a 3L earthenware pot, and 380ml of distilled water is added at a material-to-liquid ratio of 1:40 (g / ml). The mixture is soaked for 30 minutes, first brought to a boil over high heat, then simmered over low heat for 1 hour. The solution is filtered through gauze into a volumetric flask, yielding a final volume of 80ml, resulting in an extract concentration of 118.75mg / ml. After cooling, the extract is refrigerated at 4℃ for later use.

[0095] Using a sterile cotton swab, apply the prepared bacterial suspension evenly in three different directions onto an MH agar plate. Squeeze any excess suspension back into the test tube and allow to air dry at room temperature for 3-5 minutes, ensuring even adhesion and no liquid accumulation. On the dried plate, use a sterile punch to make one hole per plate vertically. After punching, use sterile forceps to remove the agar core from the well. Using a sterile pipette, precisely inject 80 μL of agarwood stem extract at a concentration of 118.75 mg / mL into the well, ensuring the well is completely filled without overflowing. Label the well after addition to avoid confusion. Incubate the plate at 35-37°C under aerobic conditions for 16-18 hours and observe the results. The results showed normal growth of Vibrio alginolyticus with no obvious inhibition zone.

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

Claims

1. Application of Aquilaria sinensis (Lour.) Spreng. leaf extract in the preparation of an antibacterial agent against Vibrio alginolyticus, wherein the aquilaria leaf extract is obtained by extracting aquilaria leaves with water by crushing and filtering, and the ratio of aquilaria leaves to water is 1g:2-4mL.

2. The application according to claim 1, characterized in that, The ratio of agarwood leaves to water is 1g:4mL.

3. The application according to claim 1 or 2, characterized in that, The preparation method of the agarwood leaf extract includes the following steps: take agarwood leaves, add water, crush into a homogenate, filter to remove impurities, centrifuge, collect the supernatant, sterilize, and obtain the agarwood leaf extract.

4. The application according to claim 3, characterized in that, The preparation method of the agarwood leaf extract includes the following specific steps: S1, adding water to fresh agarwood leaves and crushing them into a homogenate using a crusher to obtain agarwood leaf pulp; S2, filtering the agarwood leaf pulp to remove impurities to obtain agarwood leaf coarse filtrate; S3, centrifuging the coarse filtrate and collecting the agarwood leaf supernatant; S4, filtering the supernatant to obtain agarwood leaf refined extract; S5, sterilizing the agarwood leaf refined extract under high temperature and high pressure to obtain agarwood leaf extract.

5. The application according to claim 4, characterized in that, Step S1: Use a crusher to crush for 3-5 minutes.

6. The application according to claim 4, characterized in that, Step S2, the filtration is performed using gauze.

7. The application according to claim 4, characterized in that, In step S3, the centrifugation speed is 1700-2300 rpm and the centrifugation time is 8-12 minutes.

8. The application according to claim 4, characterized in that, Step S4, the filtration is performed using filter paper.

9. The application according to claim 4, characterized in that, In step S5, the sterilization temperature is 120-122℃ and the sterilization pressure is 0.21-0.23MPa.

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