Application of mignonettetree leaf water extract in inhibition of vibrio alginolyticus

Abstract

The invention provides an application of a mignonettetree leaf water extract in inhibition of vibrio alginolyticus. According to the application of the mignonettetree leaf water extracting solution in inhibiting vibrio alginolyticus, the mignonettetree leaf water extracting solution is prepared by taking mignonettetree leaves as a raw material and adding water for extraction; the mass volume ratio g / mL of the mignonettetree leaves to the water is 1: (2-4). The mignonettetree leaf water extracting solution provided by the invention can be better applied to inhibition of vibrio alginolyticus, and the prepared mignonettetree leaf water extracting solution can effectively promote wound healing, is high in healing speed and can effectively avoid infection recurrence. Compared with an organic solvent extraction method, the mignonettetree flower and leaf water extracting solution disclosed by the invention has the advantage that the use of dangerous chemicals is avoided. The preparation method provided by the invention is simple in process, simple to operate, low in cost, easy to popularize on a large scale and the like.

Description

Technical Field

[0001] The invention relates to the field of natural plant extraction, and in particular to application of a water extract of Henna leaves in inhibiting Vibrio alginolyticus. Background Art

[0002] Henna (scientific name: Lawsonia inermisL. ), the leaves, flowers, fruits and seeds of Henna can be used clinically to treat many diseases. Henna leaves have good therapeutic effects on diarrhea, dysentery, leprosy and scabies; flowers can be used to treat headaches, fever, allergies, anemia, insomnia, etc.; seeds are effective for fever, insomnia, dysentery, diarrhea and mental retardation; bark can treat spleen enlargement and stubborn skin diseases; roots can treat leprosy.

[0003] Studies have shown that the flower and leaf extracts of Henna have inhibitory effects on Staphylococcus, Streptococcus, Brucella and Salmonella, the Henna extract (Hennaquinone) has inhibitory effects on Gram-positive and Gram-negative bacteria, the water, ethanol and ethyl acetate extracts of Henna have antibacterial activity against Escherichia coli, Staphylococcus aureus, lactic acid bacteria and Pseudomonas aeruginosa, the DMSO, ethanol and ethyl acetate extracts of Henna leaves have strong activity against Escherichia coli, Staphylococcus aureus, lactic acid bacteria and drug-resistant Candida albicans, and the extract of Henna seeds has an inhibitory effect on Pseudomonas aeruginosa.

[0004] However, the extracts obtained from different parts of Henna have very different antibacterial effects on different pathogens. In order to achieve good therapeutic effects, a lot of research is needed, and a very large amount of raw materials may be needed to have an antibacterial effect. Summary of the invention

[0005] In view of this, the present invention proposes the use of the water extract of the leaves of the hydrangea in inhibiting Vibrio alginolyticus.

[0006] In recent years, the widespread use of antibiotics has led to the increased drug resistance of Vibrio alginolyticus, and its ability to spread in the environment has made it increasingly difficult to control it with traditional drugs. The inventor has conducted a lot of research and found that using the leaves of the lily of the valley as raw material can inhibit Vibrio alginolyticus with good results.

[0007] The technical solution of the present invention is achieved in this way: Effect of water extract of Houttuynia cordata leaves on inhibiting Vibrio alginolyticus ( Vibrio alginolyticus ). The Henna leaf water extract is prepared by adding water to the Henna leaf water extract using Henna leaf as raw material; the mass volume ratio of Henna leaf to water is 1:2-4 g / mL.

[0008] A further technical solution is that the method for preparing the Henna leaf water extract comprises the following steps: taking fresh Henna leaves, adding water, grinding them with a crusher, filtering and centrifuging after the grinding is completed, and sterilizing them by high pressure to obtain the Henna leaf water extract.

[0009] A further technical solution is that the method for preparing the water extract of the leaves of the herbaceous plant comprises the following specific steps: (1) taking fresh Henna leaves, adding purified water, and grinding them using a crusher to obtain Henna leaf slurry; (2) filtering the Henna leaf slurry prepared in step (1) to obtain a coarse filtrate; (3) centrifuging the crude filtrate from step (2) and collecting the supernatant; (4) filtering the supernatant of step (3) to obtain a refined extract; (5) The extract obtained in step (4) is sterilized by high pressure to obtain a water extract of the leaves of the Herba Lysimachiae.

[0010] A further technical solution is that, in step (1), a crusher is used for grinding for 1 to 5 minutes.

[0011] A further technical solution is that, in step (2), the filtering is performed using gauze.

[0012] A further technical solution is that, in step (3), the centrifugation condition is: centrifugation at a speed of 1500-2500 rpm for 5-15 minutes.

[0013] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention has conducted a large number of studies and found that the Henna leaf water extract prepared by using Henna leaf as raw material can inhibit Vibrio alginolyticus with good effect.

[0014] (2) The water extract of the leaves of the Herba Morindae provided by the present invention can be preferably used to inhibit Vibrio alginolyticus, and compared with the extraction method using organic solvents, it avoids the use of hazardous chemicals. The preparation method proposed by the present invention has the advantages of simple process, simple operation, low cost, and easy large-scale promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Before medication - photo of the turtle's left hind limb with ulceration; Figure 2 After medication (using the water extract of the leaves of the Herba Lysimachiae prepared in Example 1) - a photo of the ulcer on the left hind limb of the turtle having healed; Figure 3 Before medication—photo of the wound on the turtle’s neck; Figure 4 After medication (using the water extract of Houttuynia cordata leaves prepared in Example 1) - a photo of the turtle's neck wound having healed. DETAILED DESCRIPTION

[0016] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0017] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0018] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0019] Example 1 The preparation of the water extract of the leaves of the herbaceous plant comprises the following steps: (1) Crushing process: Put 100 g of fresh, pest-free Houttuynia cordata leaves into a crusher, add 400 mL of purified water, and crush for 1-5 min at room temperature to obtain Houttuynia cordata leaf slurry.

[0020] (2) Filtration and impurity removal: The crushed Sedum slurry is filtered through gauze to remove coarse particles and obtain a coarse filtrate.

[0021] (3) Centrifugal separation: Transfer the crude filtrate to a centrifuge tube and centrifuge at 2000 rpm for 10 min to separate the solid and liquid, and collect the supernatant.

[0022] (4) Filtration: Filter the supernatant after centrifugation with filter paper to remove residual impurities and obtain clarified Houttuynia cordata liquid, i.e., the refined extract.

[0023] (5) Sterilization: The extract is placed in a sterile container and sterilized by high pressure and high temperature (sterilization pressure is 0.22 MPa, sterilization temperature is 121°C, and sterilization time is 30 min) to obtain the water extract of the leaves of the Herba Lysimachiae.

[0024] Test example 1 Experimental method: Pick out individual colonies of the isolated and cultured Vibrio alginolyticus and prepare them into 0.5 McFarland's bacterial suspension, inoculate them into nutrient agar medium (spread plate method), and place two Oxford cups in the culture dish after inoculation, one with 200uL of 0.25mg / mL water extract of Hylocereus discolor leaves and the other with 200uL of imipenem. After standing for 3 minutes, put them into the incubator for culture.

[0025] Test object: the water extract of the leaves of the Herba Lysimachiae prepared in Example 1.

[0026] Positive control: Imipenem (antibiotic) 2 Test results (1) Antibacterial activity against Vibrio alginolyticus Table 1 Antibacterial activity of different drugs against Vibrio alginolyticus

[0027] Note: Determination of antibacterial effect: The diameter of the antibacterial zone ≥ 20 mm indicates strong antibacterial effect, indicated by "+++"; 15 mm~20 mm indicates moderate antibacterial effect, indicated by "++"; 10 mm~15 mm indicates weak antibacterial effect, indicated by "+"; <10 mm indicates "—".

[0028] The results show that the water extract of the leaves of the Herba Lysimachiae prepared in Example 1 has a strong antibacterial activity against Vibrio alginolyticus, achieving the antibacterial effect of imipenem, and can be a good alternative to antibiotics for inhibiting Vibrio alginolyticus.

[0029] The preparation of the water extract of Henna rhizome comprises the following steps: (1) Crushing process: Put 5 g of fresh, pest-free rhizomes of the herb into a crusher, add 20 mL of purified water, and crush at room temperature for 1-5 min into a homogenous slurry to obtain the rhizome slurry of the herb.

[0030] (2) Filtration and impurity removal: The crushed rhizome pulp of the Herba Lysimachiae is filtered through gauze to remove coarse particles and obtain a coarse filtrate.

[0031] (3) Centrifugal separation: Transfer the crude filtrate to a centrifuge tube and centrifuge at 2000 rpm for 10 min to separate the solid and liquid, and collect the supernatant.

[0032] (4) Filtration: Filter the supernatant after centrifugation with filter paper to remove residual impurities and obtain clear foamy rhizome fluid, that is, the refined extract.

[0033] (5) Sterilization: The extract is placed in a sterile container and sterilized by high pressure and high temperature (sterilization pressure is 0.22 MPa, sterilization temperature is 121°C, and sterilization time is 30 min) to obtain the water extract of the leaves of the Herba Lysimachiae.

[0034] Table 2 Antibacterial activity of different parts of Henna against Vibrio alginolyticus

[0035] Note: Determination of antibacterial effect: The diameter of the antibacterial zone ≥ 20 mm indicates strong antibacterial effect, indicated by "+++"; 15 mm~20 mm indicates moderate antibacterial effect, indicated by "++"; 10 mm~15 mm indicates weak antibacterial effect, indicated by "+"; <10 mm indicates "—".

[0036] The results showed that the rhizome extract of Henna had no antibacterial activity against Vibrio alginolyticus and could not replace the water extract of Henna leaf in inhibiting Vibrio alginolyticus.

[0037] (2) Minimum inhibitory concentration - test tube double dilution method Table 3 Test tube double dilution method

[0038] Note: “+” indicates bacterial growth, and “-” indicates sterile growth.

[0039] The results showed that when the concentration of the water extract of the leaves of the hydrangea was 31.25~250 mg / ml, it had a good inhibitory effect on Vibrio alginolyticus, indicating that a good inhibitory Vibrio alginolyticus drug can be prepared using less raw materials.

[0040] (3) Comparative experiment on turtle wounds before and after medication In the early stage of the experiment: 1% gentian violet solution was applied to the infected wounds of turtles. It was found that the average time for the wounds to heal was 6-12 weeks, the wound healing process was slow, the healing condition was poor, and there was a potential risk of recurrence.

[0041] 3.1 Eight turtles whose wounds were confirmed to be infected with Vibrio alginolyticus were selected for this experiment.

[0042] 3.2 Preparation of Houttuynia cordata leaf water extract: the Houttuynia cordata leaf water extract prepared in Example 1.

[0043] 3.3 Conventional veterinary supplies: sterile water, sterile tape measure for measuring wounds, multiple independent and disinfected temporary holding pools, and a water purification system to maintain appropriate seawater temperature and salinity.

[0044] 3.4 Fix the turtle and carefully disinfect the wound 3-5 times with sterile water, with the range extending 1-2 cm beyond the edge of the wound. 3.5 Use a sterile cotton swab to dip an appropriate amount of water extract of the leaves of the hydrangea plant and slowly apply it on the wound surface to ensure complete coverage. Record the time of medication and take photos. 3.6 The experiment should continue for 3-4 weeks to ensure sufficient time to observe the wound healing process. 3.7 After the experiment, all data were summarized: the average time for the turtles’ wounds to heal was 3 weeks, and no recurrence of infection was found. Figure 1-4 shown.

[0045] The results showed that the water extract of the leaves of the Herba Lysimachiae prepared in Example 1 effectively promoted the healing of turtle wounds, with a fast healing speed, and could effectively prevent the recurrence of infection.

[0046] Example 2 The main difference from Example 1 is that 200 mL of purified water is added to 100 g of Henna leaves. Other treatments are consistent with Example 1. The prepared Henna leaf water extract also has a good antibacterial effect on Vibrio alginolyticus.

[0047] Comparative Example 1 Antibacterial activity of the aqueous extract of the leaves of Sedum odoratum against other bacteria Table 4 Antibacterial activity of water extract of the leaves of the hyacinthus annuus against other bacteria

[0048] Note: Determination of antibacterial effect: The diameter of the antibacterial zone ≥ 20 mm indicates strong antibacterial effect, indicated by "+++"; 15 mm~20 mm indicates moderate antibacterial effect, indicated by "++"; 10 mm~15 mm indicates weak antibacterial effect, indicated by "+"; <10 mm indicates "—".

[0049] It can be seen that the water extract of the leaves of the hollyhock plant has a certain inhibitory effect on Staphylococcus aureus and Pseudomonas aeruginosa, but the effect is poor. It has no inhibitory effect on Escherichia coli and Enterococcus faecalis. It can be seen that the inhibitory effect of the extract from the same part on different pathogens varies greatly.

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the scope of the present invention should be included in the protection scope of the present invention.

Claims

1. The use of the water extract of the leaves of the herbaceous plant is characterized by: The water extract of the leaves of the hollyhock plant has an inhibitory effect on Vibrio alginolyticus ( Vibrio alginolyticus ), the Henna leaf water extract is prepared from Henna leaves as raw materials, and water is added to extract the Henna leaf water extract; the mass volume ratio of the Henna leaf to water (g / mL) is 1:2~4.

2. The use according to claim 1, characterized in that: The preparation method of the Henna leaf water extract comprises the following steps: taking Henna leaves, adding water, grinding them with a crusher, filtering and centrifuging after the grinding, and sterilizing them by high pressure to obtain the Henna leaf water extract.

3. The use according to claim 2, characterized in that: The preparation method of the water extract of the leaves of the hyacinthus annuus comprises the following specific steps: (1) taking fresh Henna leaves, adding purified water, and grinding them using a crusher to obtain Henna leaf slurry; (2) filtering the Henna leaf slurry prepared in step (1) to obtain a coarse filtrate; (3) centrifuging the crude filtrate from step (2) and collecting the supernatant; (4) filtering the supernatant of step (3) to obtain a refined extract; (5) The extract obtained in step (4) is sterilized by high pressure to obtain a water extract of the leaves of the Herba Lysimachiae.

4. The use according to claim 3, characterized in that: Step (1), grinding with a crusher for 1 to 5 minutes.

5. The use according to claim 3, characterized in that: Step (2), the filtering is performed using gauze.

6. The use according to claim 3, characterized in that: Step (3), the centrifugation condition: centrifugation at a speed of 1500-2500 rpm for 5-15 minutes.

7. The use according to claim 1, characterized in that: It is used to prepare medicines for treating wounds infected with Vibrio alginolyticus.

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