Preparation and application of acaricidal preparation based on ligusticum wallichii essential oil

Abstract

The invention discloses preparation and application of an acaricidal preparation based on ligusticum wallichii essential oil. The preparation method comprises a preparation method of a ligusticum wallichii essential oil solution and a preparation method of a ligusticum wallichii essential oil microemulsion. According to the preparation and application of the mite killing preparation based on the ligusticum wallichii essential oil, the killing effect of the ligusticum wallichii essential oil on sarcoptic mites is obviously related to the concentration of the essential oil after the action time of the essential oil is prolonged for 4 hours, and the mite killing effect of the ligusticum wallichii essential oil is enhanced along with the increase of the concentration of the essential oil; the corrected death rate is increased to different degrees, and when the concentration is 2.5 mg / mL, the compound also has a certain killing effect on sarcoptic mite adults.

Description

Technical Field

[0001] The invention relates to the technical field of cutting equipment, and in particular to the preparation and application of a mite-killing preparation based on chuanxiong essential oil. Background Art

[0002] Scabies is an important worldwide infectious zoonosis caused by scabies of the genus Scabies in the family Sarcoptes that parasitize the epidermis of the skin of humans and other mammals. Scabies can be divided into ordinary scabies (OS) and crusted scabies (CS) according to the degree of infection and clinical characteristics. Ordinary scabies is common in cases where the number of infected mites is small, and the main manifestations are erythematous papules and allergic skin reactions accompanied by itching. Crusted scabies has more obvious clinical manifestations than ordinary scabies, is highly contagious, and is accompanied by the risk of mite proliferation, hyperkeratotic crusting and severe secondary infection. Secondary bacterial infections mainly include Staphylococcus aureus and Streptococcus pyogenes, and may also cause secondary diseases such as pyoderma, acute glomerulitis and rheumatic heart disease, which can lead to the death of animals in severe cases.

[0003] Chuanxiong is an annual herb of the genus Ligusticum of the Apiaceae family, mainly distributed in Dujiangyan, Pengzhou, Xindu, Chongzhou and other areas of Sichuan Province, my country. It is reported that Chuanxiong essential oil plays a pharmacological role in the prevention and treatment of various diseases. At present, in the field of plant protection, there are many studies on the insecticidal and acaricidal activity of Chuanxiong essential oil, while the research on the biological activity of Chuanxiong essential oil on plant pathogenic fungi is relatively limited. In subsequent studies, the acaricidal activity of 6 plant essential oils on Tetranychus cinnabarinus was studied, and it was found that Chuanxiong essential oil had the highest activity; its LC50 (median lethal concentration) was 0.38 mg / mL.

[0004] During the development of scabies, the toxins, excretions, and dead insect bodies secreted by scabies mites can cause inflammatory reactions and allergic reactions in surrounding tissues or organs. From the above, it can be seen that Chuanxiong essential oil can inhibit the inflammatory reaction and release of inflammatory factors in animal bodies, regulate the level of antibodies in the body, reduce inflammatory cell infiltration, and has a more obvious effect in anti-inflammatory and anti-allergic reactions. It can be inferred that Chuanxiong essential oil has a certain effect on the life process of scabies mites and the pathogenic mechanism of scabies. Therefore, the following experiment was designed to identify the in vitro mite-killing effect of Chuanxiong essential oil. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a preparation and application of a mite-killing preparation based on Chuanxiong essential oil, aiming to solve the above problems.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A method for preparing a miticide preparation based on chuanxiong essential oil comprises a method for preparing a chuanxiong essential oil solution and a chuanxiong essential oil microemulsion.

[0008] Preferably, the preparation method of the Chuanxiong essential oil solution comprises:

[0009] A diluent with a ratio of glycerol to DMSO of 1:1 was prepared, and then the corresponding solid Chuanxiong essential oil was weighed and dissolved in the diluent to obtain Chuanxiong essential oil solutions with concentrations of 1, 2, 4, and 8 mg / ml.

[0010] Preferably, the preparation method of the Chuanxiong essential oil microemulsion comprises:

[0011] (1) First, prepare a mixture of surfactant and co-surfactant in a mass ratio of 2:1, stir evenly with a magnetic stirrer, and let stand for equilibrium;

[0012] (2) Add Chuanxiong essential oil in a mass ratio of 7:3 to the liquid in step (1) and stir evenly to obtain an oil phase mixture, then add water dropwise to the oil phase while stirring to obtain a clear and transparent microemulsion, and finally make the water content 60%.

[0013] Preferably, the method for preparing the Chuanxiong essential oil microemulsion uses Tween-80 as a surfactant, selects ethanol as a co-surfactant, and the Km value is 4:1-1:0, preferably 2:1.

[0014] Preferably, the final concentration of the essential oil in the Chuanxiong essential oil microemulsion is 12%, and the final characterization is that the conductivity and particle size of the essential oil are 102 mS / m and 14.4 nm, respectively.

[0015] Preferably, the preparation prepared by the preparation method according to any one of claims 1 to 6 is used for in vitro miticide.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The invention provides a preparation and application of a miticide preparation based on Chuanxiong essential oil. The killing effect of Chuanxiong essential oil on scabies increases with the extension of the action time of the essential oil. After 4 hours of action, the killing effect of Chuanxiong essential oil on scabies is significantly correlated with the concentration of the essential oil. The miticide effect is enhanced with the increase of the concentration of the essential oil, and the corrected mortality rate increases to varying degrees. When the concentration is 2.5 mg / mL, the preparation also has a certain killing effect on adult scabies.

[0018] After 8 hours of action, the killing effect of Chuanxiong essential oil on scabies mites was still significantly correlated with the concentration of essential oil, and its mite killing effect increased with the increase of essential oil concentration. 10 mg / mL Chuanxiong essential oil showed significant mite killing effect at this action time.

[0019] After 12 hours of action, the killing effect of Chuanxiong essential oil on scabies mites was similar to that at concentrations of 10 mg / mL and 5 mg / mL. After 24 hours of action, Chuanxiong essential oil showed a significant mite-killing effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the survival curve of the two kinds of plant essential oils of the present invention after acting on scabies mites at a concentration of 10 mg / mL;

[0021] Figure 2 It is a schematic diagram of the survival curve of the two plant essential oils of the present invention after acting on scabies mites at a concentration of 5 mg / mL;

[0022] Figure 3 It is a schematic diagram of the survival curve of the two plant essential oils of the present invention after acting on scabies mites at a concentration of 2.5 mg / mL. DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The experimental equipment involved in the present invention is:

[0025] Six-well cell culture dishes, dissecting needles, scalpels, constant temperature incubators, optical microscopes, alcohol lamps, and filter paper are all equipment available on the market.

[0026] The experimental materials and animals involved in the present invention are:

[0027] Chuanxiong essential oil, glycerin, dimethyl sulfoxide (DMSO), UP water, and New Zealand rabbits naturally infected with scabies can all be purchased on the market.

[0028] Example 1

[0029] This embodiment is a method for preparing a solution of Chuanxiong essential oil:

[0030] A diluent with a ratio of glycerol to DMSO of 1:1 was prepared, and then the corresponding solid Chuanxiong essential oil was weighed and dissolved in the diluent to obtain Chuanxiong essential oil solutions with concentrations of 1, 2, 4, and 8 mg / ml.

[0031] Example 2

[0032] This embodiment is a method for collecting diseased materials:

[0033] Rabbits naturally infected with scabies were euthanized, their limbs were removed, and the fur was cut off as much as possible. After exposing the affected part, the dish was placed in a cleaned and disinfected large flat dish and placed in a constant temperature incubator at 35°C. After about an hour, a large number of scabies mites could be seen crawling out of the affected skin. After morphological identification under a microscope, the dishes were collected for later use.

[0034] Example 3

[0035] This example is a mite killing activity test of Chuanxiong essential oil, comprising the following steps:

[0036] 1. The in vitro experiment was divided into a negative control group, an experimental group and a positive control group. The negative control group was a solution of glycerol and DMSO mixed in a ratio of 1:1. The experimental group was a solution of Chuanxiong essential oil of different concentrations. The positive control group was 1% ivermectin. The solutions of Chuanxiong essential oil of different concentrations were the solutions of Chuanxiong essential oil of 1, 2, 4 and 8 mg / ml in Example 1.

[0037] 2. Place a pre-prepared dry filter paper of appropriate size at the bottom of a clean six-well cell culture dish, apply glycerol to the wall to prevent scabies mites from crawling out, facilitate recording and observation, and make grouping marks;

[0038] 3. The plastic bag method was used, i.e., a diamond-shaped notch of about 3*1mm was cut with ophthalmic scissors at the upper left corner of the plastic bag (4.0x6.0cm), sealed with medical breathable adhesive tape, a small amount of vaseline was applied inside to prevent mites from sticking, and ventilation was performed. Filter paper (the edge of the filter paper was fitted with the inner edge of the plastic bag, and the size was 5.2x4.0cm) was added to the ventilated plastic bag, and about 30 adult mites in the adult stage were picked out with a mite picking needle per bag, and divided into X groups, with 3 repeated tests in each group. A solution of glycerol and DMSO 1:1 mixed and a 1% ivermectin solution were added to the negative control group and the positive control group, respectively. 200ul of 1, 2, 4, and 8mg / ml Chuanxiong essential oil solution were added to the experimental groups, respectively, and then the groups were placed at room temperature for 0.5h, 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, and 24h to record the number of survivors and deaths;

[0039] 4. Use a dissecting needle to randomly pick 30 healthy live scabies mites and place them in each well of the culture dish, cover the lid, and place in a constant temperature incubator at 25°C and 75% relative humidity for cultivation;

[0040] 5. Observe and record the survival of scabies mites under an optical microscope at certain time intervals (observe once every 15 minutes, observe once every 1 hour after 30 minutes, observe once every 2 hours after 2.5 hours, until all mites in the experimental group die), and record the number of dead mites and the time when all mites died in each observation;

[0041] 6. Repeat the experiment three times and obtain the average value.

[0042] Example 4

[0043] This example is the determination of the median lethal concentration (LC50) and median lethal time (LT50) of Chuanxiong essential oil:

[0044] After the experiment in Example 3 above proves that Chuanxiong essential oil has a killing effect on scabies in vitro, an experiment is conducted to determine the median lethal concentration (LC50) and median lethal time (LT50) of the drug. The specific operation steps include:

[0045] 1. The experiment was divided into five groups. Group I was the control group, which was DMSO and glycerol (1:1) solution. Groups II-V were 1, 2, 4, and 8 mg / ml of Chuanxiong essential oil solution.

[0046] 2. Mark the five groups in step (1) using a six-well cell culture dish, place a dry filter paper at the bottom of each well, and coat the four walls with glycerol;

[0047] 3. For the five groups in step (1), add 10 drops of the corresponding solution to each group;

[0048] 4. Use a dissecting needle to randomly select 30 healthy live scabies mites and place them in each well of the culture dish. Cover the dish with a lid and place it in a constant temperature incubator at 25°C and a relative humidity of 75%.

[0049] 5. Observe and record the death of scabies mites under an optical microscope at certain time intervals (observe once every 15 minutes, observe once every 1 hour after 30 minutes, observe once every 2 hours after 2.5 hours, until all mites in the experimental group die) until all mites die;

[0050] 6. Repeat the experiment three times.

[0051] Example 5

[0052] This embodiment is a detection index and a detection method:

[0053] The criteria for judging the death of insects are as follows: if the insect's limbs are motionless and tremble when slightly heated, it is paralysis; if the limbs of mites are motionless and there is no sign of recovery when slightly heated by a heat source, or if the insect's body shrinks, changes color, or becomes deformed, it is judged to be dead.

[0054] Under a 40×10 optical microscope, continuously observe the insect's claws or paws for one minute. If the insect does not move, use a dissecting needle to stimulate it. If the insect still does not move, it is preliminarily judged to be dead. After thirty minutes at room temperature, observe the paws again for one minute. If there is still no movement, the insect is judged to be dead.

[0055] Example 6

[0056] This example is the preparation and characterization of Chuanxiong essential oil microemulsion:

[0057] The preparation method uses Tween-80 as a surfactant and selects ethanol as a co-surfactant, with a Km value of 4:1-1:0, preferably 2:1.

[0058] 1. First, prepare a mixture of surfactant and co-surfactant in a mass ratio of 2:1, stir evenly with a magnetic stirrer, and let it stand for equilibrium;

[0059] 2. Add Chuanxiong essential oil in a mass ratio of 7:3 to the liquid in step (1) and stir evenly to obtain an oil phase mixture, then add water dropwise to the oil phase while stirring to obtain a clear and transparent microemulsion, and finally make the water content 60%.

[0060] The characterization is as follows: after preparation, the final concentration of the essential oil in the microemulsion is 12%, and finally after a series of characterizations, the conductivity and particle size of the essential oil are 102 mS / m and 14.4 nm, respectively.

[0061] Example 7

[0062] This example is the clinical application of Chuanxiong essential oil microemulsion:

[0063] The 12% essential oil microemulsion in Example 6 was used to treat sick rabbits. A total of 24 rabbits diagnosed with scabies were treated by external spray. The treatment lasted for 7 days with a cure rate of 100%. On the 7th day of medication, the affected area recovered and began to grow new hair.

[0064] Example 8

[0065] This embodiment is a data processing method:

[0066] Mortality rate (%) = 100 × number of dead insects / number of test insects

[0067] Corrected mortality rate (%) = 100 × [treatment group mortality rate - control group mortality rate / (1 - control group mortality rate)]

[0068] The data were expressed as "mean ± standard deviation" and were statistically analyzed using SPSS17.0 software. The significance analysis was performed using the t-test. The relationship between mortality and concentration or time was evaluated using two-variable correlation analysis. The probability value regression model and weighted regression analysis were used to calculate the LC50 and LT50 and their 95% confidence limits and the toxicity regression equation.

[0069] Example 9

[0070] This example is the experimental results of the above example:

[0071] 1.50% lethal concentration of 2 kinds of plant essential oils against scabies

[0072] After 4 hours of action, the LC50 of Chuanxiong essential oil and Patchouli essential oil for scabies were 5.152 mg / mL and 3.555 mg / mL, respectively, indicating that scabies were sensitive to both essential oils. Within 12 hours of action, the LC50 of the two essential oils decreased with the increase of action time; comparing the two essential oils, the LC50 of Patchouli essential oil was less than that of Chuanxiong essential oil at the same action time, as shown in Table 1.

[0073] 2. Survival curve of scabies mites under the action of different concentrations of plant essential oils

[0074] From the survival curve, it can be seen that Chuanxiong essential oil and Patchouli essential oil have good mite killing effects. With the increase of survival time, the survival rate of scabies mites at each concentration basically decreases in a step-like manner; however, when the essential oil concentration is 10 mg / mL, scabies mites die faster after 2 hours of action of Chuanxiong essential oil and Patchouli essential oil, and the curve drops steeply, as shown in Figure 1. Figures 1 to 3 .

[0075] 3.2 Contact effects of plant essential oils on scabies mites at different concentrations

[0076] The killing effects of the two plant essential oils on scabies increased with the extension of the action time of the essential oils. After 4 hours of action, the killing effects of the two plant essential oils on scabies were significantly correlated with the concentration of the essential oils (P<0.05), and the killing effect increased with the increase of the concentration of the essential oils. The 10 mg / mL concentration of Chuanxiong essential oil and Patchouli essential oil showed good killing effects, and the corrected mortality rates of scabies were 74.67% and 88.00%, respectively. The two plant essential oils also had a certain killing effect on adult scabies at a concentration of 2.5 mg / mL.

[0077] After 8 hours of action, the killing effect of Chuanxiong essential oil on scabies was still significantly correlated with the concentration of essential oil (P<0.05), and its killing effect increased with the increase of essential oil concentration, while the killing effect of Patchouli essential oil on scabies was similar at concentrations of 5mg / mL and 2.5mg / mL (P>0.05), but both showed good killing effect. 10mg / mL Chuanxiong essential oil and Patchouli essential oil showed significant killing effect at this action time, and the corrected mortality rates of scabies were 98.53% and 97.06%, respectively.

[0078] After 12 hours of action, the killing effect of Chuanxiong essential oil on scabies was similar to that at 10 mg / mL and 5 mg / mL (P>0.05), but different from the killing effect at 2.5 mg / mL (P<0.05); the killing effect of Patchouli essential oil on scabies at three concentrations was not significantly different, and P<0.05 in the variance homogeneity test. However, when the concentration of the two plant essential oils was 10 mg / mL, the corrected mortality rate of scabies reached 100%, and the killing effect was significant; at the lowest concentration of 2.5 mg / mL, it also showed an excellent killing effect.

[0079] After 24 hours of action, both plant essential oils showed significant miticide effects. Among them, when the Chuanxiong essential oil was at the lowest concentration of 2.5 mg / mL, the corrected mortality rate of scabies reached 97.06%, and Patchouli essential oil also had a good killing effect on scabies at this concentration, with a corrected mortality rate of scabies of 76.47%. See Tables 2-3.

[0080] Table 1

[0081] The median lethal concentration (LC50) of two plant essential oils against scabies

[0082] Table1 Half lethal concentration of two essential oils against Sarcoptes scabiei (LC50)

[0083]

[0084]

[0085] Note: “*” indicates a 95% confidence interval

[0086] Note: "*" means 95%confidence interval

[0087] Table 2 The contact killing effect of Chuanxiong essential oil on scabies mites at different concentrations

[0088] Table2 The contact killing effect of two kinds of Ligusticumchuanxiong essential oil at different concentrations against Sarcoptesscabiei

[0089]

[0090]

[0091] Note: The data in the table are the average values ​​of 3 replicates. Different lowercase letters after the data in the same column indicate significant differences (P<0.05).

[0092] Note:The data in the table is the average of the three repetitions.Corresponding data are marked with different lower case lettersindicate significant differences(P<0.05).The same as below.

[0093] Table 3 The contact killing effect of Patchouli essential oil on scabies at different concentrations

[0094] Table3 The contact killing effect of two kinds of Ageratum essentialoil at different concentrations against Sarcoptes scabiei

[0095]

[0096]

[0097] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0098] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing acaricide preparation based on Chuanxiong essential oil, characterized in that: The invention comprises a preparation method of a chuanxiong essential oil solution and a chuanxiong essential oil microemulsion.

2. The method for preparing a miticide preparation based on Chuanxiong essential oil according to claim 1, characterized in that: The preparation method of the chuanxiong essential oil solution comprises: A diluent with a ratio of glycerol to DMSO of 1:1 was prepared, and then the corresponding solid Chuanxiong essential oil was weighed and dissolved in the diluent to obtain Chuanxiong essential oil solutions with concentrations of 1, 2, 4, and 8 mg / ml.

3. The method for preparing a miticide preparation based on Chuanxiong essential oil according to claim 1, characterized in that: The preparation method of the chuanxiong essential oil microemulsion comprises: (1) First, prepare a mixture of surfactant and co-surfactant in a mass ratio of 2:1, stir evenly with a magnetic stirrer, and let stand for equilibrium; (2) Add Chuanxiong essential oil in a mass ratio of 7:3 to the liquid in step (1) and stir evenly to obtain an oil phase mixture, then add water dropwise to the oil phase while stirring to obtain a clear and transparent microemulsion, and finally make the water content 60%.

4. The method for preparing a miticide preparation based on Chuanxiong essential oil according to claim 3, characterized in that: The preparation method of the Chuanxiong essential oil microemulsion uses Tween-80 as a surfactant, selects ethanol as a co-surfactant, and has a Km value of 4:1-1:0, preferably 2:

1.

5. The method for preparing a miticide preparation based on Chuanxiong essential oil according to claim 3, characterized in that: The final concentration of the essential oil in the Chuanxiong essential oil microemulsion is 12%, and the final characterization is that the conductivity and particle size of the essential oil are 102 mS / m and 14.4 nm, respectively.

6. An application of a miticide preparation based on Chuanxiong essential oil, characterized in that: The invention relates to an application of a preparation prepared by the preparation method according to any one of claims 1 to 6 for killing mites in vitro.

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