Application of aqueous extract of Lawsonia inermis leaves in inhibiting Vibrio alginolyticus

Abstract

The present invention provides an application of the aqueous extract of Lawsonia inermis leaves in inhibiting Vibrio alginolyticus. The aqueous extract of Lawsonia inermis leaves of the present invention is used in inhibiting Vibrio alginolyticus ( Vibrio alginolyticus ), and the aqueous extract of Lawsonia inermis leaves is prepared by adding water to Lawsonia inermis leaves as raw materials for extraction; the mass-volume ratio of Lawsonia inermis leaves to water is 1:2-4 g / mL. The aqueous extract of Lawsonia inermis leaves provided by the present invention can be preferably used in inhibiting Vibrio alginolyticus. The prepared aqueous extract of Lawsonia inermis leaves of the present invention can effectively promote wound healing with a fast healing speed, and can effectively avoid the recurrence of infection. Moreover, compared with the extraction method using organic solvents, the aqueous extract of Lawsonia inermis leaves of the present invention avoids the use of chemical dangerous goods. The preparation method proposed by the present invention has a simple process, is easy to operate, has a low cost, and is easy to be popularized on a large scale, etc.

Description

Technical Field

[0001] The present invention relates to the field of natural plant extraction, and particularly to the application of the aqueous extract of Lawsonia inermis leaves in inhibiting Vibrio alginolyticus. Background Art

[0002] Lawsonia inermis (scientific name: Lawsonia inermis L. ) The leaves, flowers, fruits, and seeds of Lawsonia inermis can be used to treat many diseases clinically. The leaves of Lawsonia inermis have good curative effects on diarrhea, dysentery, leprosy, and scabies; the flowers can be used to treat headache, fever, allergy, anemia, insomnia, etc.; the seeds are effective against fever, insomnia, dysentery, diarrhea, and mental retardation; the bark can treat splenomegaly and skin stubborn diseases; the roots can treat leprosy.

[0003] Studies have shown that the extracts of the flowers and leaves of Lawsonia inermis have inhibitory effects on Staphylococcus, Streptococcus, Brucella, and Salmonella. The extract of Lawsonia inermis (lawsone) has inhibitory effects on Gram-positive bacteria and Gram-negative bacteria. The aqueous, ethanol, and ethyl acetate extracts of Lawsonia inermis have antibacterial activities against Escherichia coli, Staphylococcus aureus, Lactobacillus, and Pseudomonas aeruginosa. The DMSO, ethanol, and ethyl acetate extracts of Lawsonia inermis leaves have strong activities against Escherichia coli, Staphylococcus aureus, Lactobacillus, and drug-resistant Candida albicans. The extract of Lawsonia inermis seeds has an inhibitory effect on Pseudomonas aeruginosa.

[0004] However, the extracts obtained from different parts of Lawsonia inermis have very significant differences in the antibacterial effects against different pathogens. To obtain good curative effects, a large amount of research is required, and a very large amount of raw materials may be needed to have antibacterial effects. Summary of the Invention

[0005] In view of this, the present invention proposes the application of the aqueous extract of Lawsonia inermis leaves in inhibiting Vibrio alginolyticus.

[0006] In recent years, the widespread use of antibiotics has led to an increase in the drug resistance of Vibrio alginolyticus. Coupled with its transmission ability in the environment, it has become increasingly difficult to control with traditional drugs. The inventor has conducted a large amount of research and found that using Lawsonia inermis leaves as raw materials can inhibit Vibrio alginolyticus, and the effect is good.

[0007] The technical solution of the present invention is realized as follows:

[0008] The aqueous extract of Lawsonia inermis leaves is applied in inhibiting Vibrio alginolyticus ( Vibrio alginolyticus ). The aqueous extract of Lawsonia inermis leaves is prepared by using Lawsonia inermis leaves as raw materials and adding water for extraction; the mass-volume ratio of Lawsonia inermis leaves to water is 1:2 - 4 g / mL.

[0009] A further technical solution is that the preparation method of the Lawsonia inermis leaf water extract comprises the following steps: taking fresh Lawsonia inermis leaves, adding water, grinding with a crusher, filtering, centrifuging after grinding is completed, and performing high-pressure sterilization treatment to obtain the Lawsonia inermis leaf water extract.

[0010] A further technical solution is that the preparation method of the Lawsonia inermis leaf water extract comprises the following specific steps:

[0011] (1) Taking fresh Lawsonia inermis leaves, adding purified water, and grinding with a crusher to obtain Lawsonia inermis leaf pulp;

[0012] (2) Filtering the Lawsonia inermis leaf pulp in step (1) to obtain a crude filtrate;

[0013] (3) Centrifuging the crude filtrate in step (2) and collecting the supernatant;

[0014] (4) Filtering the supernatant in step (3) to obtain a refined extract;

[0015] (5) Performing high-pressure sterilization treatment on the refined extract in step (4) to obtain the Lawsonia inermis leaf water extract.

[0016] A further technical solution is that in step (1), grinding is performed with a crusher for 1 to 5 minutes.

[0017] A further technical solution is that in step (2), the filtering is performed using a gauze.

[0018] A further technical solution is that in step (3), the centrifugation conditions are: centrifuging at a speed of 1500 to 2500 revolutions per minute for 5 to 15 minutes.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] (1) Through a large number of studies in the present invention, it is found that using Lawsonia inermis leaves as raw materials to prepare the Lawsonia inermis leaf water extract can inhibit Vibrio alginolyticus, and the effect is good.

[0021] (2) The Lawsonia inermis leaf water extract provided by the present invention can be preferably applied to inhibit Vibrio alginolyticus. Compared with the extraction method using organic solvents, it avoids the use of chemical dangerous goods. The preparation method proposed by the present invention has a simple process, is easy to operate, has a low cost, and is easy to be popularized on a large scale, etc. Description of the Drawings

[0022] Figure 1 Photo before medication - photo of the ulcer on the left hind limb of the sea turtle;

[0023] Figure 2 Photo after medication (using the Lawsonia inermis leaf water extract prepared in Example 1) - photo of the healed ulcer on the left hind limb of the sea turtle;

[0024] Figure 3 Before medication - photo of the wound on the turtle's neck;

[0025] Figure 4 After medication (using the aqueous extract of Lawsonia inermis leaves prepared in Example 1) - photo of the completely healed wound on the turtle's neck. Detailed implementation manners

[0026] To better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0027] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0028] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can all be obtained from commercial channels.

[0029] Example 1

[0030] The preparation of the aqueous extract of Lawsonia inermis leaves includes the following steps:

[0031] (1) Crushing treatment: Put 100 g of fresh, pest - and - disease - free Lawsonia inermis leaves into a crusher, add 400 mL of purified water, and crush at room temperature for 1 - 5 min to form a homogenate, obtaining Lawsonia inermis leaf pulp.

[0032] (2) Filtration to remove impurities: Filter the crushed Lawsonia inermis leaf pulp with gauze to remove coarse - grained residues, obtaining a crude filtrate.

[0033] (3) Centrifugal separation: Transfer the crude filtrate to a centrifuge tube, centrifuge at a speed of 2000 revolutions per minute for 10 min to separate the solid from the liquid, and collect the supernatant.

[0034] (4) Filtration: Filter the supernatant after centrifugation with filter paper to remove residual impurities, obtaining a clear Lawsonia inermis liquid, that is, the refined extract.

[0035] (5) Sterilization treatment: Fill the refined extract into a sterile container, and perform high - pressure and high - temperature sterilization (sterilization pressure is 0.22 MPa, sterilization temperature is 121 °C, sterilization time is 30 min) to obtain the aqueous extract of Lawsonia inermis leaves.

[0036] Test examples

[0037] 1 Test method: Pick single colonies from the isolated and cultured Vibrio alginolyticus and prepare a bacterial suspension of 0.5 McFarland respectively, inoculate it in a nutrient agar medium (plate spreading method). After inoculation, place 2 Oxford cups in the petri dish, add 200 μL of the aqueous extract of Lawsonia inermis leaves with a concentration of 0.25 mg / mL to one cup and 200 μL of imipenem to the other cup. After standing for 3 min, put it into an incubator for cultivation.

[0038] Test object: The aqueous extract of Lawsonia inermis leaves prepared in Example 1.

[0039] Positive control: Imipenem (antibiotic)

[0040] 2 Test results

[0041] (1) Bacteriostatic activity against Vibrio alginolyticus

[0042] Table 1 Bacteriostatic activity of different drugs against Vibrio alginolyticus

[0043]

[0044] Note: Judgment of bacteriostatic effect: A bacteriostatic zone diameter ≥20 mm indicates strong bacteriostatic effect, denoted by "+++"; 15 mm - 20 mm indicates moderate bacteriostatic effect, denoted by "++"; 10 mm - 15 mm indicates weak bacteriostatic effect, denoted by "+"; <10 mm is denoted by "—".

[0045] The results show that the aqueous extract of Lawsonia inermis leaves prepared in Example 1 has strong bacteriostatic activity against Vibrio alginolyticus, achieving the bacteriostatic effect of imipenem, and can preferably replace antibiotics for inhibiting Vibrio alginolyticus.

[0046] Preparation of the aqueous extract of Lawsonia inermis rhizome includes the following steps:

[0047] (1) Crushing treatment: Put 5 g of fresh, pest - and - disease - free Lawsonia inermis rhizome into a crusher, add 20 mL of purified water, and crush at room temperature for 1 - 5 min to form a homogenate, obtaining Lawsonia inermis rhizome pulp.

[0048] (2) Filtration to remove impurities: Filter the crushed Lawsonia inermis rhizome pulp with gauze to remove coarse particle residues, obtaining a crude filtrate.

[0049] (3) Centrifugal separation: Transfer the crude filtrate to a centrifuge tube, centrifuge at a speed of 2000 revolutions per minute for 10 min to separate the solid from the liquid, and collect the supernatant.

[0050] (4) Filtration: Filter the centrifuged supernatant with filter paper to remove residual impurities, obtaining a clear Lawsonia inermis rhizome liquid, i.e., the refined extract.

[0051] (5) Sterilization treatment: Put the refined extract into a sterile container and sterilize it by high - pressure and high - temperature (sterilization pressure is 0.22 MPa, sterilization temperature is 121 °C, sterilization time is 30 min) to obtain the aqueous extract of Lawsonia inermis leaves.

[0052] Table 2 Bacteriostatic activity of different parts of Lawsonia inermis against Vibrio alginolyticus

[0053]

[0054] Note: Judgment of bacteriostatic effect: When the diameter of the bacteriostatic circle is ≥20 mm, it indicates strong bacteriostatic effect, denoted by "+++"; when it is 15 mm - 20 mm, it indicates moderate bacteriostatic effect, denoted by "++"; when it is 10 mm - 15 mm, it indicates weak bacteriostatic effect, denoted by "+"; when it is <10 mm, it is denoted by "—".

[0055] The results showed that the extract of Lawsonia inermis rhizome had no bacteriostatic activity against Vibrio alginolyticus and could not replace the water extract of Lawsonia inermis leaves in inhibiting Vibrio alginolyticus.

[0056] (2)Minimum inhibitory concentration - tube dilution method

[0057] Table 3 Tube dilution method

[0058]

[0059] Note: "+" indicates the growth of bacteria, and "—" indicates no growth of bacteria.

[0060] The results showed that when the concentration of the water extract of Lawsonia inermis leaves was 31.25 - 250 mg / ml, it had a good inhibitory effect on Vibrio alginolyticus, indicating that a good drug for inhibiting Vibrio alginolyticus could be prepared with less raw materials.

[0061] (3)Comparison experiment before and after applying medicine to the wounds of sea turtles

[0062] Before the experiment: The infected wounds of sea turtles were smeared with 1% methyl violet solution. It was found that the average wound healing time of sea turtles was 6 - 12 weeks, the wound healing process was slow, the healing condition was poor, and there was a potential risk of recurrence.

[0063] 3.1 A total of 8 sea turtles with wounds infected by Vibrio alginolyticus confirmed by detection were selected in this experiment.

[0064] 3.2 Preparation of the water extract of Lawsonia inermis leaves: The water extract of Lawsonia inermis leaves prepared in Example 1.

[0065] 3.3 Conventional veterinary supplies: Sterile water, a sterile soft ruler for measuring wounds, multiple independent and disinfected temporary holding ponds, a system for maintaining appropriate seawater temperature, salinity and water purification.

[0066] 3.4 Fix the sea turtle and carefully disinfect the wound with sterile water 3 - 5 times, with the scope exceeding 1 - 2 cm beyond the wound edge.

[0067] 3.5 Use a sterile cotton swab to dip an appropriate amount of the water extract of Lawsonia inermis leaves and slowly smear it on the wound surface to ensure complete coverage, record the medication time and take pictures.

[0068] 3.6 Continue the experiment for 3 - 4 weeks to ensure sufficient time to observe the wound healing process.

[0069] 3.7 After the experiment, all data were summarized: the average wound healing time of the sea turtles was 3 weeks, and no recurrence of infection was found. As Figures 1-4 shown.

[0070] The results showed that the aqueous extract of Lawsonia inermis leaves prepared in Example 1 effectively promoted the wound healing of sea turtles, with a fast healing speed, and could effectively avoid the recurrence of infection.

[0071] Example 2

[0072] The main difference from Example 1 was that 100 g of Lawsonia inermis leaves were added with 200 mL of purified water. Other treatments were the same as those in Example 1. The prepared aqueous extract of Lawsonia inermis leaves also had good antibacterial effects against Vibrio alginolyticus.

[0073] Comparative Example 1

[0074] Antibacterial activity of the aqueous extract of Lawsonia inermis leaves against other bacteria

[0075] Table 4 Antibacterial activity of the aqueous extract of Lawsonia inermis leaves against other bacteria

[0076]

[0077] Note: Judgment of antibacterial effect: an antibacterial zone diameter ≥20 mm indicates strong antibacterial effect, represented by "+++"; 15 mm - 20 mm indicates moderate antibacterial effect, represented by "++"; 10 mm - 15 mm indicates weak antibacterial effect, represented by "+"; <10 mm is represented by "-".

[0078] Thus, it can be seen that the aqueous extract of Lawsonia inermis leaves has certain inhibitory effects on Staphylococcus aureus and Pseudomonas aeruginosa, but the effects are not good, and it has no inhibitory effect on Escherichia coli and Enterococcus faecalis. It can be seen that the inhibitory effects of the extract from the same part on different pathogens vary greatly.

[0079] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the scope of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of aqueous extract of Lawsonia inermis leaves, characterized in that, The aqueous extract of Lawsonia inermis leaves is used in the preparation of an antibacterial agent against Vibrio alginolyticus ( Vibrio alginolyticus ), and the aqueous extract of Lawsonia inermis leaves is prepared by using Lawsonia inermis leaves as a raw material and adding water for extraction; the mass-volume ratio of Lawsonia inermis leaves to water is 1:2-4 g / mL. The preparation method of the Lawsonia inermis leaf water extract comprises the following specific steps: (1) Take fresh Lawsonia inermis leaves, add purified water, and grind them using a crusher to obtain Lawsonia inermis leaf pulp; (2) Filter the Lawsonia inermis leaf pulp obtained in step (1) to obtain a crude filtrate; (3) Centrifuge the crude filtrate obtained in step (2) and collect the supernatant; (4) Filter the supernatant obtained in step (3) to obtain a refined extract; (5) Sterilize the refined extract obtained in step (4) by high-pressure sterilization to obtain the Lawsonia inermis leaf water extract; In step (1), grind for 1 to 5 minutes using a crusher; In step (3), the centrifugation conditions are: centrifuge at a speed of 1500 to 2500 revolutions per minute for 5 to 15 minutes.

2. The application according to claim 1, wherein In step (2), filter using gauze.

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