Application of Badong buddleia lindleyana alcohol extract in preparation of mosquito repellent

Abstract

The invention belongs to the technical field of mosquito repelling, and particularly relates to application of Badong buddleia alcohol extract in preparation of a mosquito repelling agent. The alcohol extract of Badong buddleia has a repelling effect on aedes albopictus, the Badong buddleia Soxhlet alcohol extract with the concentration of 0.2 g / mL has the best repelling effect on aedes albopictus, the effective protection rate can reach 95.83%, and the Badong buddleia Soxhlet alcohol extract can be used for preparing the mosquito repelling agent.

Description

Technical Field

[0001] This invention belongs to the field of mosquito repellency technology, specifically relating to the application of Buddleja officinalis alcohol extract in the preparation of mosquito repellents. Background Technology

[0002] Aedes albopictus ( Aedes albopictus Aedes albopictus is a native mosquito species to Asia. Starting in the 1980s, with the increasing global trade, it began to spread globally and was selected by the World Health Organization as a highly dispersive mosquito species. Aedes albopictus is distributed in many parts of my country, can breed in various environments, and often inhabits dark, damp places. It is a highly effective vector for viral transmission. Related studies have shown that Aedes albopictus is mainly transmitted through natural infection or experiments and can transmit more than 40 viruses, including dengue fever, Zika virus disease, malaria, yellow fever, chikungunya, and Japanese encephalitis. These infectious diseases cause serious harm to humans. For example, Aedes albopictus is an important vector for Zika virus, and infection can cause microcephaly, meningitis, and other serious neurological diseases in newborns. In recent years, especially during the hot summer months, Aedes albopictus mosquitoes have begun to be active on a large scale. Various related diseases caused by Aedes albopictus mosquitoes have been introduced from overseas, increasing the risk of infectious disease outbreaks and gradually causing outbreaks across the country. This seriously threatens human health and causes irreversible damage to the human body, which has attracted great attention from local governments.

[0003] Currently, common mosquito repellents on the market include N,N-Diethyl-3-methyl benzoyl amide (DEET), Picaridin (also known as Icaridin, KBR3023, or Bayrepel™), and Icaridin (IR3535). DEET is a widely used mosquito repellent; although it effectively prevents mosquito bites, long-term or excessive use may cause skin irritation, such as redness, swelling, and itching, and may also lead to central nervous system effects or poisoning. Furthermore, DEET may pollute the environment; therefore, it is recommended to follow the product instructions and use it under the guidance of a doctor. Picaridin, as a highly effective and slightly toxic mosquito repellent, exhibits excellent long-lasting and broad-spectrum insecticidal effects, but a small number of people may experience skin allergic reactions (such as redness and itching) after use, although the incidence is lower than with traditional DEET. DEET has a longer-lasting and more effective mosquito-repellent effect, and is safer than N,N-diethyl-3-ethyl (DEET). It is less toxic and less irritating, and more environmentally friendly, but it has the potential to penetrate the skin and cause neurological and skin diseases. In summary, existing mosquito-repellent substances have side effects. The effective ingredients in natural plant-based repellents come from plant extracts. Developing natural plant-based mosquito repellent products can significantly reduce the side effects on humans and the threat to the environment. However, to date, only a few products use natural ingredients, such as lemon balm leaf oil, citronella oil, and catnip oil. Whether the extract of Buddleja officinalis has a mosquito-repellent effect is still unknown. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides the application of *Buddleja officinalis* alcohol extract in the preparation of mosquito repellents.

[0005] Application of Buddleja officinalis alcohol extract in mosquito repellents. This invention discovers that the alcohol extract of Buddleja officinalis has a repellent effect on mosquitoes, and therefore proposes its application in mosquito repellents.

[0006] Preferably, the mosquito includes Aedes albopictus.

[0007] Preferably, the preparation steps of the Buddleja officinalis ethanol extract are as follows: Buddleja officinalis ethanol is extracted with ethanol to obtain the Buddleja officinalis ethanol extract; The ratio of the herb *Buddleja officinalis* to the ethanol is 1g:10mL to 30mL.

[0008] Preferably, when extracting Buddleja officinalis with ethanol, Soxhlet extraction is used.

[0009] Preferably, when using ethanol to extract *Buddleja officinalis*, the extraction method is used, in which the *Buddleja officinalis* is soaked in the ethanol for extraction.

[0010] Preferably, when extracting *Buddleja officinalis* using ethanol, the *Buddleja officinalis* is added to the ethanol and then extracted using ultrasound at 45℃~50℃. The main components of *Buddleja officinalis* include volatile terpenes, flavonoids, and phenylethyl glycosides, all of which have certain mosquito-repellent effects. Specifically: volatile terpenes (such as linalool and α-pinene) volatilize significantly at temperatures above 50℃, and the extraction rate is very low below 40℃; flavonoids (such as apigenin and luteolin) decompose significantly above 60℃; phenylethyl glycosides (such as verbascoside) are extracted at 40℃~50℃, as they are quite sensitive to heat, and high temperatures easily cause the glycosidic bonds to break.

[0011] Preferably, the mosquito repellent is composed of the Buddleja officinalis alcohol extract and a mosquito repellent-acceptable carrier.

[0012] Preferably, the carrier of the mosquito repellent is selected from at least one of water, ethanol, propylene glycol, jojoba oil, gelatin, gum arabic, Tween 80, glyceryl stearate, glyceryl tricaprylate, cetyl alcohol, lanolin, urea, petrolatum, stearyl alcohol, beeswax, and polyoxyethylene oleyl alcohol ether.

[0013] Preferably, when the acceptable carrier is ethanol, the ratio of the Buddleja officinalis extract to the ethanol is 0.03g to 0.25g: 1mL.

[0014] Preferably, the mosquito repellent is an oil-based, alcohol-based, or water-based formulation.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention found that the alcohol extract of Buddleja officinalis has a repellent effect on Aedes albopictus, and the 0.2 g / mL concentration of Soxhlet's alcohol extract of Buddleja officinalis has the best repellent effect on Aedes albopictus, with an effective protection rate of up to 95.83%, which can be developed as a natural plant-derived repellent. Attached Figure Description

[0016] Figure 1 Ethanol extraction rate of Buddleja officinalis under three different extraction methods.

[0017] Figure 2 Analysis of the mean effective protection rate of the same extract at different concentrations.

[0018] Figure 3 Analysis of the mean effective protection rate of different extracts at the same concentration. Detailed Implementation

[0019] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0020] 1. Experimental Materials The experimental animal material for this invention—Aedes albopictus—was collected at 103°45′0″E, 29°33′34″N, at an altitude of 353.04 m. Under suitable conditions (25–33°C), the time from hatching of Aedes albopictus eggs to emergence of adult mosquitoes was 10.5–11.5 days. To simulate the natural environment, buckets containing small amounts of dead leaves and tap water were placed in bamboo groves and weed beds, respectively, and collected after about a week. The laboratory temperature was maintained at 24°C–28°C and the relative humidity at 60% to ensure similarity to the natural breeding environment of Aedes albopictus. After emergence, microscopic examination confirmed the mosquito's identity as Aedes albopictus. The mosquitoes were then fed with 0.1 g / mL glucose solution and starved for 12 hours before the experiment.

[0021] The *Buddleja officinalis* used in this invention was collected from a location at 10°44′56″E, 29°33′54″N, at an altitude of 350.59 meters.

[0022] Anhydrous ethanol (Chengdu Kelong Chemicals Co., Ltd.), Soxhlet extractor BSXT-06-150 (Shanghai Bilang Instrument Manufacturing Co., Ltd.), ultrasonic processor FS-1200 (Shanghai Shengxi Ultrasonic Instrument Co., Ltd.), rotary evaporator RE-2000B (Shanghai Yarong Biochemical Instrument Factory), forced-air drying oven WGLL-125BE (Tianjin Tester Instrument Co., Ltd.), cyclone pulverizer FS-11 (Zhejiang Top Cloud Agriculture Technology Co., Ltd.), precision electronic balance FA-2004 (Shanghai Jingke Balance Instrument Factory), medical refrigerator HYC-940 (Qingdao Haier Biomedical Co., Ltd.).

[0023] 2. Test methods (1) Plant extracts Take leaves of *Buddleja officinalis* and dry them in a 60℃ electric thermostatic drying oven until constant weight. Grind the dried leaves into powder using a pulverizer, then pass the resulting plant powder through a 60-mesh sieve. Finally, place the sieved powder in a sealed bag for later use.

[0024] 1) Soxhlet extraction method Weigh 2g of *Buddleja officinalis* powder, wrap it in filter paper, and place it in the siphon tube of a Soxhlet extractor. Add 20mL of anhydrous ethanol to a volumetric flask and extract at 83℃. Once the solution in the siphon tube turns colorless, collect the solution in the volumetric flask. Set the rotary evaporator to 83℃ and 20 rpm. After the extract has concentrated to a paste and all ethanol has evaporated, weigh it again and transfer the extract to a brown wide-mouth bottle, labeling it. Store the brown wide-mouth bottle in a 4℃ refrigerator for later use.

[0025] 2) Extraction method Weigh 2g of *Buddleja officinalis* powder, soak it in 8mL of anhydrous ethanol, and place it in a dark place. Stir it three times a day, and after 24 hours, filter it. Then add 6mL of anhydrous ethanol to the filter residue for extraction. This step is called extraction one.

[0026] Perform extraction steps two and three in the same manner as in step one.

[0027] The residues from extractions one, two, and three were rinsed three times with 18 ml of ethanol, and then the filtrates from extractions one, two, and three were combined.

[0028] The obtained filtrate was concentrated to a paste using a rotary evaporator, and weighed after the ethanol had completely evaporated. The rotary evaporator was set up under the same conditions as in the Soxhlet extraction method. The extract was then placed into brown wide-mouth bottles and labeled. The brown wide-mouth bottles were stored in a refrigerator at 4°C for later use.

[0029] 3) Ultrasonic extraction method Weigh 2g of *Buddleja officinalis* powder into an Erlenmeyer flask, add 10mL of anhydrous ethanol, sonicate for 10min (50℃, 70% ultrasonic power), and then filter. Add 6mL of anhydrous ethanol to the residue, sonicate for 10min, and filter. Add another 4mL of anhydrous ethanol to the residue, sonicate for 10min, filter the liquid, and finally combine all the filtrates.

[0030] Repeat the above experiment three times. Concentrate the solution to a paste using a rotary evaporator. After the ethanol has completely evaporated, weigh the solution. Then, pack the extract into a brown wide-mouth bottle and label it. Finally, place the brown wide-mouth bottle in a refrigerator at 4°C for later use.

[0031] Calculate the plant extraction rate using the following formula: R represents the plant component extraction rate, W represents the weight of the dried plant powder (g), and W1 represents the weight of the plant extract (g). (2) Human attack experiments Referencing the National Standard of the People's Republic of China, Indoor Efficacy Tests and Evaluation of Sanitary Insecticides for Pesticide Registration, Part 9: Test Methods for Repellents (GB / T 13917.9-2009).

[0032] Expose 4cm x 4cm of skin on the back of your hand (cover the rest), without applying any medication to the exposed skin. If more than 20 mosquitoes are found within two minutes, the mosquito attack capability is considered acceptable.

[0033] (3) Human approach-avoidance test 600 test mosquitoes were placed in a mosquito cage that was 40cm long, 30cm wide, and 30cm high.

[0034] First, prepare the extract of Buddleja officinalis in anhydrous ethanol to concentrations of 0.2 g / mL, 0.1 g / mL, and 0.05 g / mL.

[0035] After passing the attack power test, a 4cm x 4cm area of ​​skin was exposed on the back of the hand. The treatment group reagent was applied to this area at a dose of 1.5 μL / cm². The blank control reagent was applied to the other hand, and the rest of the hand was completely covered. After waiting for 5 minutes, the hand was placed into the cage, and the number of mosquitoes that landed on it within 2 minutes was counted. After 2 minutes, the hand was removed, and after waiting for 15 minutes, the test was repeated. This process was repeated 5 times, and the entire procedure was repeated 3 times. Before each repetition, a human attack test was conducted again to ensure that the mosquitoes' attack power was up to standard. A repellency rate greater than 80% was considered to have a good mosquito-repelling effect.

[0036] Formula for calculating repellency (effective protection rate) %: Nc2 represents the number of mosquito bites in the control group, and Nt2 represents the number of mosquito bites in the treatment group. Data processing and analysis The raw data were processed and plotted using Excel 2021. SPSS R26.0.0.0 was used to perform significance and correlation analysis on the effective protection rate of Buddleja officinalis extract against Aedes albopictus.

[0037] Results and Analysis 1. Analysis of extraction rates obtained from three different extraction methods Using three different extraction methods—immersion extraction, ultrasonic extraction, and Soxhlet extraction—the extraction rates were as follows: Figure 1 As shown. From Figure 1As can be seen, under the same material-to-liquid ratio, the extraction rates of the three extraction methods were 0.18%, 0.34%, and 0.43%, respectively, and there were significant differences in the extraction rates among the three methods. Soxhlet extraction showed the best extraction rate, significantly higher than the other two methods.

[0038] 2. Repellent effect of extracts obtained by different extraction methods against Aedes albopictus mosquitoes. The results of the repellency effects of different extraction methods of Buddleja officinalis alcohol extract on Aedes albopictus are shown in Table 1. Among them, the Soxhlet extract showed better repellency, and the 0.2 g / mL Soxhlet extract was particularly effective.

[0039] The Soxhlet extract, formulated at a concentration of 0.2 g / mL, showed the highest repellency rate against Aedes albopictus, reaching 95.83%, and provided effective protection throughout the test period, with an average repellency rate of 93.06%. Repellency rates decreased at concentrations of 0.1 g / mL and 0.05 g / mL, but both still provided effective protection. The 0.1 g / mL and 0.2 g / mL extracts provided effective protection, while the 0.05 g / mL extract provided no protection. The 0.2 g / mL ultrasonic extract provided effective protection against Aedes albopictus, while the 0.1 g / mL and 0.05 g / mL extracts provided no protection. Anhydrous ethanol provided no protection throughout the test period and had a low effective protection rate.

[0040] Table 1. Repellent effect of Buddleja officinalis extract on Aedes albopictus mosquitoes. Table 2 shows the effective protection rates of the Soxhlet extract of *Buddleja officinalis* obtained by Soxhlet extraction, prepared at concentrations of 0.2 g / mL, 0.1 g / mL, and 0.05 g / mL. As shown in Table 2, in five repeated tests, the effective protection rates of the 0.1 g / mL and 0.2 g / mL Soxhlet extracts showed no significant difference in repellency, and both concentrations maintained effective protection. This indicates that the repellency effects of the 0.1 g / mL and 0.2 g / mL Soxhlet extracts were relatively stable. However, the effective protection rate of the 0.05 g / mL Soxhlet extract showed significant differences between the first test and the subsequent four tests, as well as between the second and third tests and the last two tests. This indicates that the repellency effect decreased significantly over time.

[0041] In the first and second tests, there were no significant longitudinal differences among different concentrations of Soxhlet extract. However, in the third, fourth, and fifth tests, the differences between the 0.2 g / mL and 0.1 g / mL concentrations of Soxhlet extract and the 0.05 g / mL concentration were significant, indicating that the repulsive effect of the 0.05 g / mL concentration of Soxhlet extract was significantly lower than that of the 0.1 g / mL and 0.2 g / mL concentrations. Overall, the repulsive effect of the 0.05 g / mL concentration of Soxhlet extract was unstable and showed a clear downward trend, while the repulsive effects of the 0.1 g / mL and 0.2 g / mL concentrations showed a stable downward trend.

[0042] Table 2. Analysis of the effective protection rate of Soxhlet extract at different dilution concentrations Note: Different lowercase letters in the same row indicate significant differences at the 0.05 level; different uppercase letters in the same column indicate significant differences at the 0.05 level.

[0043] Table 3 shows the effective protection rates of the *Buddleja officinalis* ethanol extract obtained by ultrasonic extraction and diluted to concentrations of 0.2 g / mL, 0.1 g / mL, and 0.05 g / mL. Table 3 shows that the 0.2 g / mL extract showed no significant difference in repellency between the first three tests and the fifth test, and no significant difference between the fourth and fifth tests, indicating that the repellency effect was relatively stable in the first four tests, but decreased from the fourth test onwards. The 0.1 g / mL extract showed a significant difference in repellency between the first test and the last four tests, but no significant difference between the last four tests, indicating that the repellency effect decreased significantly from the second test onwards, and the downward trend eventually leveled off. For the 0.05 g / mL ultrasonic extract, the repellency effects differed significantly between the first, second, and last two tests, but not significantly between the second and third tests, indicating that the repellency effect decreased in a stepwise manner and did not provide effective protection.

[0044] Longitudinal significance analysis of the repulsion effect of ultrasonic extracts at different concentrations showed that in the first four tests, the repulsion effect of 0.2 g / mL concentration was significantly different from that of 0.1 g / mL and 0.05 g / mL concentrations. In the last test, the repulsion effect of 0.2 g / mL concentration was significantly different from that of 0.05 g / mL concentration. This indicates that in the first four tests, the repulsion effect of 0.2 g / mL concentration was better than that of 0.05 g / mL and 0.1 g / mL concentrations. In the last test, the difference in repulsion effect between 0.2 g / mL concentration and 0.1 g / mL concentration decreased, indicating that the repulsion effect of 0.2 g / mL concentration was significantly reduced.

[0045] Table 3. Analysis of the effective protection rate of ultrasonic extracts at different dilution concentrations. Note: Different lowercase letters in the same row indicate significant differences at the 0.05 level; different uppercase letters in the same column indicate significant differences at the 0.05 level.

[0046] Table 4 presents the effective protection rates of the ethanol extract of Buddleja officinalis obtained by the immersion method, diluted to concentrations of 0.2 g / mL, 0.1 g / mL, and 0.05 g / mL, respectively, in experiments. As shown in Table 4, the avoidance effect was not significant in the five tests at the three concentrations, indicating that the avoidance effect did not decrease significantly over time and that the avoidance effect was relatively stable.

[0047] Longitudinal significance analysis of the repellency effects of extracts at different concentrations revealed that, based on studies of the relationship between concentration and biological effects, the first test showed little difference in repellency effects, indicating that the initial concentration had no significant impact on the repellency effect. In subsequent tests, the repellency effect at a concentration of 0.2 g / mL was not significantly different from that at 0.1 g / mL, but showed a significant repellency effect compared to 0.05 g / mL. The repellency effect at a concentration of 0.1 g / mL was also not significantly different from that at 0.05 g / mL, indicating that during the testing period, the repellency effect at a concentration of 0.2 g / mL was relatively better, while the repellency effect at a concentration of 0.1 g / mL was somewhere in between.

[0048] Table 4. Analysis of the effective protection rate of extracts at different dilution concentrations Note: Different lowercase letters in the same row indicate significant differences at the 0.05 level; different uppercase letters in the same column indicate significant differences at the 0.05 level.

[0049] Table 5 shows the effective protection rates of extracts obtained by different extraction methods during the test at a concentration of 0.2 g / mL. According to the data in Table 5, the repellency effects of the three extraction methods did not differ significantly in the first three tests, but the repellency effect of the ultrasonic extract decreased more significantly over time. For example, in the third test, the repellency effects of the Soxhlet extract and the ultrasonic extract differed significantly, and in the fifth test, the repellency effects of the immersion extract and the ultrasonic extract differed significantly. This indicates that the repellency effect of the ultrasonic extract is less stable, while the repellency effects of the Soxhlet extract and the immersion extract are relatively more stable.

[0050] Table 5. Analysis of the effective protection rate of different extraction methods at a concentration of 0.2 g / mL Note: Different lowercase letters in the same row indicate significant differences at the 0.05 level; different uppercase letters in the same column indicate significant differences at the 0.05 level.

[0051] Table 6 shows the effective protection rate of extracts obtained by different extraction methods at a concentration of 0.1 g / mL within the test time. As shown in Table 6, in the first test, the repellency effects of the Soxhlet extract at 0.1 g / mL and the immersion extract at 0.1 g / mL were significant. In the last four tests, the repellency effects of the Soxhlet extract, the ultrasonic extract, and the immersion extract were significant, indicating that the ultrasonic extract had a poorer repellency effect than the other two methods.

[0052] Table 6. Analysis of the effective protection rate of different extraction methods at a concentration of 0.1 g / mL Note: Different lowercase letters in the same row indicate significant differences at the 0.05 level; different uppercase letters in the same column indicate significant differences at the 0.05 level.

[0053] Table 7 shows the effective protection rates of extracts obtained by different extraction methods at a concentration of 0.05 g / mL within the test time. As shown in Table 7, the repellency effects of the Soxhlet extract, the maceration extract, and the ultrasonic extract differed significantly in the first two tests; in the third, fifth, and fourth tests, the repellency effects of the maceration extract and the ultrasonic extract differed significantly. Overall, the repellency effect of the Soxhlet extract decreased more significantly.

[0054] Table 7. Analysis of the effective protection rate of different extraction methods at a concentration of 0.05 g / mL Note: Different lowercase letters in the same row indicate significant differences at the 0.05 level; different uppercase letters in the same column indicate significant differences at the 0.05 level.

[0055] 3. Analysis of the average effective protection rate during the test period like Figure 2The figure shows the average effective protection rate of the same extract at different concentrations. By analyzing the significance of the effective protection rates at the three concentrations of the Soxhlet extract, maceration extract, and ultrasonic extract, it can be observed that the difference between the 0.2 g / mL and 0.05 g / mL Soxhlet extracts is significant; the difference between the 0.2 g / mL ultrasonic extract and the 0.1 g / mL and 0.05 g / mL concentrations is significant, while the others are not significant. However, the repellency effect of all three extracts decreases with decreasing concentration (0.2 g / mL, 0.1 g / mL, 0.05 g / mL). According to the standard, an effective protection rate greater than 80% indicates a repellency effect, while a rate less than 80% is considered ineffective. Therefore, it can be concluded that all three concentrations of the Soxhlet extract have effective protective effects; the 0.2 g / mL and 0.1 g / mL concentrations of the maceration extract have effective protective effects; and the 0.2 g / mL concentration of the ultrasonic extract has effective protective effects. Overall, the avoidance effect was 0.2 g / mL > 0.1 g / mL > 0.05 g / mL.

[0056] like Figure 3 The graph shows the mean effective protection rate of different extracts at the same concentration. By analyzing the significance of the effective protection rates of Soxhlet extraction, immersion extraction, and ultrasonic extraction at three concentrations, it can be observed that at concentrations of 0.05 g / mL and 0.1 g / mL, the repellent effects of Soxhlet extraction, immersion extraction, and ultrasonic extraction on Aedes albopictus are significantly different. At higher or lower concentrations, the mean values ​​of these extraction methods decrease, and the repellent effect weakens accordingly. Specifically, Soxhlet extraction, due to its simplicity and reliability, shows the best repellent effect at lower concentrations. While ultrasonic extraction has high extraction efficiency, its effect may be inferior to Soxhlet extraction at certain concentrations due to the destruction of active ingredients.

[0057] 4. Correlation analysis of time and effective protection rate Based on the analysis results in Table 8, the correlation analysis of the repellency effect of extracts obtained by test time, Soxhlet extraction, ultrasonic extraction, and immersion extraction revealed that the effective protection rate was negatively correlated with the extraction time, indicating that the repellency effect of the extract gradually decreased over time.

[0058] At a concentration of 0.2 g / mL, the repulsive effect of Soxhlet extract showed a significant correlation with time; at a concentration of 0.05 g / mL, the correlation was extremely significant, indicating that the repulsive effect at these two concentrations was unstable and decreased significantly. However, at a concentration of 0.1 g / mL, the correlation with time was not significant, suggesting that the repulsive effect at this concentration was more stable.

[0059] The three concentrations of the ultrasonic extract showed significant correlations with time, indicating that its repulsion effect was unstable and decreased significantly over time.

[0060] At concentrations of 0.1 g / mL and 0.05 g / mL, the repulsion effect of the extract showed a significant correlation with time, indicating that the repulsion effect at these concentrations was unstable. However, at a concentration of 0.2 g / mL, the repulsion effect did not show a significant correlation with time, indicating that the repulsion effect was more stable at this concentration.

[0061] Overall, the repulsive effect of Soxhlet extract at a concentration of 0.1 g / mL and that of immersion extract at a concentration of 0.2 g / mL were more stable. The repulsive effects of Soxhlet extract at a concentration of 0.2 g / mL, immersion extract at a concentration of 0.1 g / mL, and immersion extract at concentrations of 0.1 g / mL and 0.05 g / mL were the next most significant. The changes over time were not significant in the other cases.

[0062] Table 8. Correlation analysis of time, Soxhlet test, ultrasound, and extract repulsion effects. Note: * indicates a significant difference (P < 0.05), and ** indicates an extremely significant difference (P < 0.001).

[0063] Badong Buddleja officinalis ( Buddlejaalbiflora *Buddleja officinalis* (Hemsl.) is a shrub belonging to the genus *Buddleja* in the family Loganiaceae. It is widely distributed in southern my country, including Sichuan, Yunnan, and Guizhou provinces, and also found in Japan, Africa, and Malaysia. *Buddleja officinalis* grows wild in low hills, along waterways, and at forest edges, making it an excellent ornamental plant. Currently, research on the repellent effects of *Buddleja officinalis* alcohol extracts on mosquitoes is almost nonexistent. This invention utilizes *Buddleja officinalis* to investigate the repellent effect of its plant extracts on *Aedes albopictus*, aiming to provide theoretical support for developing *Buddleja officinalis* into an effective natural plant-derived repellent and lay the foundation for subsequent research.

[0064] This invention investigated the repellent effects of three different extraction methods—Soxhlet extraction, ultrasonic extraction, and maceration—and extracts at concentrations of 0.2 g / mL, 0.1 g / mL, and 0.05 g / mL on Aedes albopictus. The repellent effects of the ethanol extract of Buddleja officinalis on Aedes albopictus, from highest to lowest, were 0.2 g / mL, 0.1 g / mL, and 0.05 g / mL.

[0065] This invention found that Soxhlet extraction yielded the highest extraction rate, followed by ultrasonic extraction and maceration extraction. The experimental results showed that Soxhlet extraction had the best repellent effect, indicating a positive correlation between extraction rate and repellency rate. The repellent effect of different extraction methods decreased over time, due to factors including absorption through human skin, hydrolysis and volatilization under the influence of sweat, moisture, and microorganisms, as well as the influence of external factors such as temperature and humidity. This study demonstrates that the alcoholic extract of *Buddleja officinalis* has a repellent effect on *Aedes albopictus*, with the 0.2 g / mL concentration of Soxhlet's alcoholic extract showing the best repellent effect, achieving an effective protection rate of 95.83%. It can be considered a promising natural plant-derived repellent.

[0066] It should be noted that, in the claims of this invention, when numerical ranges are involved, it should be understood that both endpoints of the numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention only describes preferred embodiments.

[0067] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0068] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from the spirit and scope of the invention. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Application of Buddleja officinalis alcohol extract in the preparation of mosquito repellents.

2. The application according to claim 1, characterized in that, The mosquitoes mentioned include Aedes albopictus.

3. The application according to claim 1, characterized in that, The preparation steps of the Buddleja officinalis ethanol extract are as follows: Buddleja officinalis ethanol is extracted with ethanol to obtain the Buddleja officinalis ethanol extract; The ratio of the herb *Buddleja officinalis* to the ethanol is 1g:10-30mL.

4. The application according to claim 3, characterized in that, When extracting Buddleja officinalis from Badong using ethanol, the Soxhlet extraction method is employed.

5. The application according to claim 3, characterized in that, When extracting *Buddleja officinalis* from Badong using ethanol, the *Buddleja officinalis* is extracted by soaking it in the ethanol using an immersion extraction method.

6. The application according to claim 3, characterized in that, When extracting *Buddleja officinalis* from Badong using ethanol, the *Buddleja officinalis* is added to the ethanol and then extracted using ultrasound at 45℃~50℃.

7. The application according to claim 1, characterized in that, The mosquito repellent is composed of the Buddleja officinalis alcohol extract and a mosquito repellent-acceptable carrier.

8. The application according to claim 7, characterized in that, The mosquito repellent can be carried by at least one of the following: water, ethanol, propylene glycol, jojoba oil, gelatin, gum arabic, Tween 80, glyceryl stearate, glyceryl tricaprylate, cetyl alcohol, lanolin, urea, petrolatum, stearyl alcohol, beeswax, and polyoxyethylene oleyl alcohol ether.

9. The application according to claim 8, characterized in that, When the acceptable carrier is ethanol, the ratio of the Buddleja officinalis extract to the ethanol is 0.03g to 0.25g: 1mL.

10. The application according to claim 1, characterized in that, The mosquito repellent is an oil-based, alcohol-based, or water-based formulation.

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