Chinese herbal medicine composite additive and application of Chinese herbal medicine composite additive as antidote to insecticide poisoning resistance of bees
Patent Information
- Application Number
- CN202411566356.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-05
- Publication Date
- 2025-01-28
AI Technical Summary
Bees are poisoned by mistakenly consuming ethrin insecticides such as deltamethrin, resulting in high mortality rates, and the prior art is difficult to effectively detoxify and defend.
The Chinese herbal medicine composite additives include resveratrol, vitamin C, tea polyphenols, and slab blue root. As a detoxification agent, they are fed to bees to improve their antivilation and immunity.
The survival rate of bee is significantly increased after eating pesticides of chrysanthemums, reducing the mortality, and improving the health of bees, including improving food consumption, learning memory and crawling ability, and enhancing the activity of antioxidant enzymes and detoxification enzymes.
Smart Images
Figure CN119367414A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of antidotes, and in particular to a Chinese herbal medicine composite additive and an application thereof as an antidote for bees to resist insecticide poisoning. Background Art
[0002] As one of the most important pollinating insects in the world, the economic, social and ecological benefits of bees are of great and far-reaching significance to agricultural production and the maintenance of biodiversity in ecosystems. They are an indispensable and important part of ecosystems and have made important contributions to the ecological balance of nature. At the same time, bees have become a model organism for studying social insects because of their complex social structure and characteristics. Bees play a vital role in human life, so conducting scientific research on bees and improving their protection are currently hot issues around the world.
[0003] Pyrethroid insecticides, including pyrethroids, have a strong contact effect on insects. Some varieties also have stomach poison or fumigation effects, but none of them have systemic effects. Its mechanism of action is mainly to disrupt the normal physiology of insect nerves, causing them to die from excitement, spasm to paralysis. This type of insecticide affects the normal function of the insect nervous system, causing the insect to be unable to move normally and eventually die. Specifically, the mechanism of action of pyrethroids is to disrupt the permeability of insect nerve membranes, inhibit the conduction of insect nerve axons, and destroy neuronal function by interacting with sodium ion channels, causing poisoned insects to be overexcited, paralyzed and die. Deltamethrin is a highly effective neurotoxic pyrethroid insecticide that mainly acts on ion channel proteins. It is currently the most toxic of pyrethroid insecticides, a neurotoxin, and a commonly used field insecticide in modern agriculture. This insecticide is highly toxic to Italian honey bees. According to my country's pesticide toxicity classification standards for bees, deltamethrin is poisoning. Summary of the invention
[0004] The purpose of the present invention is to provide a Chinese herbal medicine composite additive and its application as an antidote for bees to resist insecticide poisoning.
[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:
[0006] In a first aspect, a Chinese herbal medicine compound additive is provided. The components of the Chinese herbal medicine compound additive include, by weight: 5 parts of resveratrol, 100 parts of vitamin C, 1 part of tea polyphenols, and 100 parts of isatis root.
[0007] Furthermore, the concentration of resveratrol is 0.05 g / L.
[0008] Furthermore, the concentration of tea polyphenols is 0.01 g / L.
[0009] In a second aspect, a Chinese herbal medicine composite additive is provided for use in the preparation of an antidote for pyrethroid insecticides.
[0010] The beneficial effects of the present invention are:
[0011] 1. The present invention first discovered that the Chinese herbal compound additive can significantly improve the survival rate of bees that accidentally ingested the pyrethroid insecticide deltamethrin and reduce their mortality rate. The experimental results of the present invention show that the mortality rate of bees that accidentally ingested pyrethroid insecticides was greatly reduced after using the Chinese herbal compound additive, and the bees after detoxification were also very healthy; at the same time, the food consumption of poisoned bees increased after feeding with the Chinese herbal compound additive; the learning, memory and crawling abilities of bees were significantly increased after feeding with the Chinese herbal compound additive, and the activities of detoxification enzymes and antioxidant enzymes were increased. The intervention of the Chinese herbal compound additive can improve the degree of insecticide poisoning of apparently healthy bees and help the normal honey collection of bees.
[0012] 2. The present invention provides a new function of Chinese herbal compound additives for bee pyrethroid insecticide infection and provides a basis for the study of bee immune defense mechanism. The Chinese herbal compound additives can be used to prepare drugs or feeds for controlling insecticides or improving bee immunity, providing a new method for controlling insecticides and having good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 Schematic diagram of the analysis of the survival and mortality rates of healthy and poisoned bees after intervention with Chinese herbal compound additives;
[0014] Figure 2 This is a schematic diagram of the analysis of food consumption after the Chinese herbal compound additives intervened in the poisoned bees; the squares of different colors on the right represent the number of exposure days;
[0015] Figure 3 This is the effect of adding Chinese herbal compound additives on the sucrose responsiveness and learning and memory of honey bees fed with deltamethrin. The horizontal axis of the sucrose responsiveness graph represents different sucrose concentrations; in the learning and memory graph, T represents the learning process, and L represents the memory process;
[0016] Figure 4 Schematic diagram of the effect of Chinese herbal compound additive intervention on the crawling ability of honey bees;
[0017] Figure 5 Schematic diagram of the effect of Chinese herbal compound additive intervention on honey bee enzyme activity. DETAILED DESCRIPTION
[0018] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.
[0019] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.
[0020] Example 1 Intervention of healthy bees and poisoned bees using Chinese herbal medicine composite additives
[0021] 1. Test samples
[0022] Italian honey bees were obtained from the apiary of the Chinese Apiculture Research Institute (N39♀59035.3300, E116♀11059.7400). The test bees were healthy and had not been exposed to pesticides before the experiment.
[0023] 2. Preparation of syrup
[0024] The preparation and feeding of syrup were adjusted according to the 2016 standard for feeding Apis mellifera larvae (Karl Crailsheim, Robert Brodschneider, Pierrick Aupinel, Dieter Behrens, Elke Genersch, Jutta Vollmann & Ulrike Riessberger-Gallé. Standard methods for artificial rearing of Apis mellifera larvae. Journal of Apicultural Research, 2013, 52(1): 1-15.).
[0025] 3. Preparation of Chinese herbal compound additives
[0026] Calculated by weight, the components of the Chinese herbal medicine composite additive include: 5 parts of resveratrol, 100 parts of vitamin C, 1 part of tea polyphenols, and 100 parts of isatis root.
[0027] Use sterilized ultrapure water to prepare a 1g / l stock solution for later use. Prepare a mixed Chinese herbal medicine additive according to the compound ratio, and dilute it with bee feed according to the needs of each age to obtain the experimental concentration of the Chinese herbal medicine compound additive during the test.
[0028] 4. Preparation of pyrethroid insecticide deltamethrin
[0029] Use sterilized ultrapure water to prepare a 1 mg / ml stock solution for later use. During the test, dilute it with bee feed according to the age of the bees, and the feeding concentration of cypermethrin is the LC10 after 48 hours of acute exposure.
[0030] 5. Sucrose sensitivity behavioral test
[0031] On the 14th day of rearing, 24 newly released bees were randomly selected from each treatment group for behavioral testing of beak extension reaction. After the test bees were starved for 4 hours, they were fixed in a hollow plastic tube to ensure that their bodies were fixed, their heads were exposed outside the tube and they could move freely. First, the sensitivity of the bees to different concentrations of sucrose was tested: a pointed cotton swab filled with 0.3%, 1%, %, 10%, and 30% sucrose solution was used to approach the bee antennae for 3 seconds respectively. The test recorded whether each bee would actively issue a beak extension reflex. Each test concentration was separated by 2 minutes. During the test, the bees did not actually consume sugar water. After the test, the bees were given 1 μl of 30% sugar water.
[0032] 6. Honey bee learning and memory behavior test
[0033] After the sensitivity test of bees to sucrose solutions of different concentrations, the bees were tested for learning and memory behavior. Since the normal sensitivity of bees to sucrose solutions is the basis for testing learning and memory ability, those bees that did not have a proboscis reflex to any concentration of sucrose solution were eliminated from the learning and memory behavior test. Then the learning and memory behavior test was conducted. First, odor learning was conducted. Linalool was dipped into a cotton swab and contacted with the bee antennae for 3 seconds, and the proboscis reaction was recorded. The test was divided into two stages: (1) Conditioned stimulus stage (CS): Keep the bees in contact with linalool for 3 seconds, and give the bees sucrose solution as a reward at the 3rd second. (2) Unconditioned stimulus stage (US): Keep the main airflow and odor stimulus airflow open for 3 seconds, without sucrose solution as a reward, and observe whether the bees actively have a proboscis reflex.
[0034] Learning ability test: After each conditioned stimulus is completed, the bees are given an unconditioned stimulus test to check whether they actively have a beak extension reflex. The number of times each bee uses the conditioned stimulus when it first has a beak extension reflex is recorded.
[0035] Memory test: After the bees repeated the learning ability test three times, they were tested again with unconditioned stimulation to check whether they actively extended their beaks and recorded. After each memory test, the fixed bees were put back into the incubator and fed a small amount of sucrose solution every hour to maintain their normal physiological response.
[0036] 7. Bee crawling ability test
[0037] Based on the phototaxis of bees, a homemade crawling device was used to measure the crawling ability of bees. That is, under the guidance of a light source, the time it took to crawl from the dark bottom of the device to the top was recorded.
[0038] 8. Changes in enzyme activity in bees
[0039] Twelve bees were randomly selected for each treatment, frozen with liquid nitrogen, and the tissues were quickly ground into powder in a mortar, transferred to a 1.5 ml centrifuge tube, added with physiological saline, centrifuged at 2500 rpm and 4°C for 10 min in a refrigerated high-speed centrifuge, and the supernatant was taken to prepare a crude enzyme solution, which was placed on ice for testing. The protein content was determined by the BCA protein concentration detection method, and the measurement of SOD, POD, CAT, and GSH activities was measured according to the instructions of the Nanjing Jiancheng kit.
[0040] 9. Experiment
[0041] A capped comb that was about to emerge from the hive was extracted from a healthy bee colony with strong colony strength, no obvious symptoms of disease and no pesticide pollution, and placed in a queen-limited egg-laying frame, and cultured in a constant temperature incubator. After culturing in the incubator for 24 hours, a sufficient number of young bees that had just emerged from the hive were collected and randomly divided into 4 groups for two experiments of acute exposure and chronic exposure; the 4 groups in both experiments were divided into a control group, a pesticide group (a group treated with cypermethrin), a Chinese herbal medicine group (a group treated with a Chinese herbal compound additive) and a detoxification group (a group treated with cypermethrin solution + a group treated with a Chinese herbal compound additive). In the acute exposure experiment, 30 samples were included in each group and repeated three times: the control group was fed only with sucrose solution for 8 days; the pesticide group and the detoxification group were fed with cypermethrin one day before, and then fed with 50% sugar water sucrose solution and Chinese herbal compound additives for 7 days; the Chinese herbal medicine group was fed with 50% sugar water sucrose solution one day before, and then fed with Chinese herbal compound additives for 7 days; in the chronic exposure experiment, 30 samples were included in each group and repeated three times: the control group was fed only with sucrose solution for 14 days; the pesticide group and the detoxification group were fed with cypermethrin for the first 7 days, and then fed with 50% sugar water sucrose solution and Chinese herbal compound additives after 7 days of treatment; the Chinese herbal medicine group was fed with 50% sugar water sucrose solution for the first 7 days, and then fed with Chinese herbal compound additives after 7 days of treatment. After exposure for 14 days, the feeding conditions and mortality of the bees were observed and recorded, and the learning and memory abilities and crawling abilities of the bees were tested.
[0042] Example 2 Analysis of the survival rate and mortality rate of healthy bees and poisoned bees after intervention with Chinese herbal compound additives
[0043] like Figure 1As shown, the survival rate of bees in the control group fed with sugar water and the Chinese herbal medicine group fed with Chinese herbal medicine compound additives can reach more than 90%, indicating that the intervention of Chinese herbal medicine compound additives will not cause abnormal death of healthy bees and will not affect the growth and development of healthy bees. The results show that the intervention of Chinese herbal medicine compound additives can prevent the infected bees from dying of disease and prevent the poisoned bee colonies from collapsing, playing an important role in the recovery and reproduction of the bee colonies.
[0044] The mortality rate of each group of bees in Example 1 was statistically analyzed. The method for determining the mortality rate of bees was as follows: the number of bee deaths on the day of pre-culture and infection was not included in the analysis to exclude the influence of mechanical death of bees during the experiment. Starting from the day after infection, the number of bee deaths was counted regularly every day, and the dead bees were removed, and the above steps were repeated until the 14th day of age.
[0045] Daily bee mortality rate = number of bees that die each day / number of bees that survive each day × 100%;
[0046] The mortality rate of bees in the whole process = the number of bee deaths / total number of samples × 100%.
[0047] The results showed that the daily mortality rate of bees in the control group fed with conventional feeding was very low. Compared with the control group, the daily mortality rate of bees after intervention with Chinese herbal compound additives did not change significantly, indicating that the intervention with Chinese herbal compound additives did not affect the growth and development of bees and could be used as auxiliary feeding for bees. In the acute exposure experiment, the mortality rate of the pesticide group and the detoxification group increased significantly. After acute exposure to insecticides for 24 hours, about 25% of the experimental bees died. After that, the mortality rate of the pesticide group continued to rise for 7 days, while the mortality rate of the detoxification group increased by less than 8%, with significant differences. In the chronic exposure experiment: the mortality rate of bees increased significantly after being treated with insecticides. Only about 50% of the experimental bees survived successfully at the age of 7 days. Compared with the treatment group, the mortality rate of bees at each age after intervention with Chinese herbal compound additives decreased significantly, and the survival rate of bees reached more than 70% at the age of 7 days. The results of the two groups of experiments showed that the intervention with Chinese herbal compound additives could significantly reduce the mortality rate of poisoned bees, prevent the infected bee colonies from collapse, and play an important role in the recovery and reproduction of bee colonies.
[0048] Example 3: Food consumption of bees after intervention with Chinese herbal compound additives
[0049] The bees in each treatment group in Example 1 were tested. Figure 2As shown in the results, the food consumption of bees in the treatment group without intervention of Chinese herbal compound additives was greatly reduced. This shows that the bees infected by the insecticides produced a toxic reaction in their bodies, and the food consumption of bees increased more or less in both acute and chronic exposure experiments after the intervention of Chinese herbal compound additives, indicating that feeding Chinese herbal compound additives alleviated the toxicity of insecticides to bees, thereby improving the appetite of bees.
[0050] Example 4 Effects of Chinese herbal compound additive intervention on sucrose sensitivity and olfactory learning and memory behavior of honey bees
[0051] In order to analyze the effect of Chinese herbal compound additive intervention on the olfactory memory and learning memory of honey bees, different concentrations of sucrose were used to test the bees in each group in Example 1. Figure 3 As shown, in both acute and chronic experiments, the beak extension rate of the pesticide group was significantly lower than that of the detoxification group; at the same time, there was almost no difference in the learning and memory results between the control group and the Chinese herbal medicine group. The results show that the Chinese herbal medicine compound additive has obvious antibacterial and antiviral effects, can effectively remove free radicals and toxins in the body; protect cells from free radicals, have good anti-stress effects, and alleviate the decline in production performance caused by stress.
[0052] Example 5 Effect of Chinese herbal compound additive intervention on the crawling ability of honey bees
[0053] like Figure 4 As shown, in the acute exposure experiment, the crawling time of the detoxification group and the Chinese herbal medicine group was significantly lower than that of the pesticide group and was almost the same as that of the control group, while in the chronic exposure experiment, the crawling time of the detoxification group was significantly lower than that of the pesticide group. The results of the two experiments showed that the Chinese herbal compound additive can reduce the crawling time of bees and improve the damage after feeding cypermethrin.
[0054] Example 6 Effect of Chinese herbal compound additive intervention on honey bee enzyme activity
[0055] like Figure 5 As shown, in the two experiments, compared with the control group, the enzyme activity of the Chinese herbal medicine group increased significantly, which indicates that after feeding the Chinese herbal medicine to the bees, the activity of the antioxidant enzymes and detoxification enzymes in the bees was activated; compared with the control group, except for the acute toxic GSH enzyme and the chronic exposure SOD enzyme, the enzyme activities of the other enzymes in the pesticide group decreased, which indicates that cypermethrin harmed the antioxidant and detoxification mechanism in the bees, and the enzyme activity of the detoxification group was slightly higher than that of the pesticide group, which indicates that the Chinese herbal medicine compound additive plays a certain antioxidant and detoxification role in the bees.
[0056] Resveratrol is a natural polyphenol. According to its role in plants, it can be classified as a low molecular weight compound of the plant antibiotic class, which is a phytochemical with antibacterial and antioxidant functions. As a polyphenol compound, resveratrol is known to have strong antioxidant activity and can be used to prevent and treat many diseases and cancers. Under oxidative stress, the body produces a large number of superoxide free radicals and can also produce superoxide dismutase through normal metabolic pathways. Resveratrol plays an antioxidant role by scavenging free radicals and regulating the activity of related enzymes. A large amount of research data confirms that resveratrol has the functions of anti-oxidative stress, anti-inflammatory, antibacterial, protecting the cardiovascular system, and regulating fat deposition. Tea polyphenols is a general term for polyphenols in tea, including catechins, anthocyanins, flavonoids, flavonols and phenolic acids. Among them, catechin compounds are the main components of tea polyphenols, which have strong antibacterial and antiviral effects and have inhibitory activity against Gram-negative bacteria, Gram-positive bacteria, DNA viruses and RNA viruses. Tea polyphenols are widely used in food chemical, medical and health industries due to their non-toxic side effects, health-care functions and non-irritating properties. In addition, some researchers have found that it is a small molecule that can extend the lifespan of a variety of organisms and protect the intestinal health of animals in animal husbandry. Vitamin C (also known as ascorbic acid) is an important water-soluble vitamin with a variety of health benefits and effects. It can not only act as a powerful antioxidant, but also neutralize free radicals in the body and reduce the damage of oxidative stress to cells. This helps slow down the aging process. It can also enhance the function of the immune system and help resist foreign infections and diseases. Isatis root is a traditional Chinese medicinal material, also known as indigo root, blue indigo root, and big indigo root. Research in natural medicinal chemistry has confirmed that isatis root contains a variety of biologically active ingredients, such as polysaccharides, polypeptides, organic acids, glucosinolates, sulfur-containing compounds, alkaloids, phospholipids, lignans, flavonoids and steroids. Modern pharmacology research has also given a more precise explanation for the efficacy of isatis root, including antipyretic, anti-inflammatory, immunity-enhancing, antiviral, anti-endotoxin and anti-cancer effects. Given the excellent antiviral and anti-inflammatory activity of isatis root, it is widely used in the prevention and treatment of viral infectious diseases and most inflammations.
[0057] According to the results of literature research, there is no report in the prior art on the use of resveratrol or tea polyphenols as an effective antioxidant active ingredient in the antidote for the pyrethroid insecticide deltamethrin. The Chinese herbal compound additive provided by this application can improve the body's immunity, improve digestion and absorption rate, and promote animal growth. On the other hand, the Chinese herbal compound additive also has obvious antioxidant and antiviral effects, which can effectively remove free radicals and toxins in the body; protect cells from free radicals, promote metabolism, have good anti-stress effects, and alleviate the decline in production performance caused by stress. Therefore, it can be used to prevent and treat bee insecticide poisoning.
[0058] The present invention takes bees treated with the pyrethroid insecticide deltamethrin as the research object, utilizes the sensitivity and learning conditions of bees to sugar water of different concentrations, carries out memory detection on the expression of Chinese herbal compound additives in the developmental stages of insecticide poisoning and healthy honey bees, as well as the increase in food consumption after feeding the Chinese herbal compound additives, and clarifies the detoxification ability of the Chinese herbal compound additives; the intervention of the Chinese herbal compound additives strongly resists the harm of insecticides to bees, reduces the mortality rate of bees, and forms a rapid and effective defense mechanism, which is used to quickly kill or eliminate exogenous pathogenic microorganisms and protect the normal honey collection of bees.
[0059] The Chinese herbal medicine composite additive provided by the present invention can significantly improve the survival rate of bees under the harm of pyrethroid pesticides, reduce their mortality rate, inhibit the toxicity of pesticides in bees, and increase the activities of antioxidant enzymes, detoxification enzymes, etc.; at the same time, it can also improve the learning and memory, crawling ability and food intake of bees. This Chinese herbal medicine composite additive can be used for bees that accidentally ingest pyrethroid pesticides remaining in pollen, which is of great significance to the beekeeping industry and has good market application value.
[0060] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.
[0061] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.
Claims
1. A Chinese herbal medicine composite additive, characterized in that: Calculated by weight, the components of the Chinese herbal medicine composite additive include: 5 parts of resveratrol, 100 parts of vitamin C, 1 part of tea polyphenols, and 100 parts of isatis root.
2. The Chinese herbal medicine composite additive according to claim 1, characterized in that: The concentration of resveratrol is 0.05 g / L.
3. The Chinese herbal medicine composite additive according to claim 1, characterized in that: The concentration of the tea polyphenols is 0.01 g / L.
4. Use of the Chinese herbal medicine composite additive according to claim 1 in the preparation of an antidote for pyrethroid insecticides.
5. The use according to claim 4, characterized in that: The pyrethroid insecticide is deltamethrin.