CN116672335B

CN116672335B - Application of tea polyphenol as antidote in bee pesticide poisoning resistance

Info

Publication number: CN116672335B
Application number: CN202310915971.8A
Authority: CN
Inventors: 高晶; 刘琳琳
Original assignee: Institute of Apicultural Research of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Apicultural Research of Chinese Academy of Agricultural Sciences
Priority date: 2023-07-25
Filing date: 2023-07-25
Publication date: 2024-03-12
Anticipated expiration: 2043-07-25
Also published as: CN116672335A

Abstract

The invention discloses an application of tea polyphenol as an antidote in bee anti-pesticide poisoning, which can relieve the toxicity of a neonicotinoid pesticide to bees. Tea polyphenols (teapolyphenols) are the general term for polyphenols in tea leaves, and include catechins, anthocyanins, flavonoids, flavonols, phenolics, etc. On one hand, the tea polyphenol can improve the learning ability, olfactory memory and crawling ability of bees, and on the other hand, the tea polyphenol can remarkably improve the survival rate and food consumption of bees and reduce toxin accumulation in the bees. The tea polyphenol has the antibacterial and antivirus effects, so that the tea polyphenol is hopeful to become a novel bioactive molecule for preventing and controlling pesticides, and a novel thought is provided for prevention and control research of infectious toxicity. The application of the tea polyphenol disclosed by the invention as an antidote in the anti-pesticide poisoning of bees can be used for relieving the harm of a neonicotinoid pesticide to bees, and has an obvious detoxification effect.

Description

Application of tea polyphenol as antidote in bee pesticide poisoning resistance

Technical Field

The invention relates to the technical field of antidotes, in particular to application of tea polyphenol as an antidote in bee pesticide poisoning resistance.

Background

The bees play a very important role in the whole ecological system. The bee pollinates the plants, so that the genetic diversity of flowering plants is ensured, the ecological balance is maintained, the diversity of plant types is maintained, and the yield and quality of crops are improved; the bees also provide bee products such as honey, propolis, royal jelly and the like, and provide basic raw materials for various industrial and civil products and the like, thereby ensuring the health of the national people.

New nicotinic insecticides are widely used since the market of the 80 s of the 20 th century, act on nicotinic acetylcholine receptors in the central nervous system of insects, cause death of insects by effectively blocking their normal neurotransmission, are the most widely used insecticide species worldwide, registering more than 120 countries. The traditional neonicotinoid insecticide has serious harm to the bee population, not only remains in the bee brain for a long time and influences the learning ability and the acquisition activity of the bee, but also reduces the survivability of the bee and shortens the service life of the bee. So research on how to alleviate the harm of the neonicotinoid insecticides to bees is urgent.

Tea polyphenols are the general names of polyphenols in tea, and comprise catechins, anthocyanins, flavonoids, flavonols, phenolic acids and the like, wherein the catechins are main components of the tea polyphenols, have strong antibacterial and disinfectant effects, and have inhibitory activity on gram-negative bacteria, gram-positive bacteria, DNA viruses and RNA viruses. The tea polyphenol has no toxic or side effect, has the characteristics of health care function, no stimulation to organisms and the like, and is widely applied to industries such as food chemical industry, medical treatment and health. The tea polyphenol has the antibacterial and antivirus effects, so that the tea polyphenol is hopeful to become a novel bioactive molecule for preventing and treating diseases, and a novel thought is provided for prevention and control research of infectious diseases.

Disclosure of Invention

The invention aims to provide an application of tea polyphenol as an antidote in bee pesticide poisoning resistance. The tea polyphenol can obviously improve the survival rate of bees under the harm of the neonicotinoid insecticides and reduce the death rate of the bees. Tea polyphenols can inhibit pesticide toxicity in bee body, and reduce activities such as antioxidant enzyme and detoxication enzyme; meanwhile, the learning and memory, crawling ability and food intake of bees can be improved. The tea polyphenol can be used for eating bees with anabasine pesticide remained in pollen by mistake, has important significance for bee-keeping industry and has good market application value.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

in a first aspect, there is provided the use of tea polyphenols or its homologues in the manufacture of a medicament for the treatment or prophylaxis of bee poisoning.

In a second aspect, there is provided the use of tea polyphenols or its homologues in the manufacture of a feed for the treatment or prophylaxis of bee poisoning.

In a third aspect, there is provided the use of tea polyphenols or its homologues for inhibiting the proliferation of pathogenic fungi of bees.

In a fourth aspect, there is provided the use of tea polyphenols or its homologues in the manufacture of an antidote for a neonicotinoid insecticide.

In a fifth aspect, there is provided the use of tea polyphenols or its homologues in the manufacture of a medicament for increasing the detoxification capacity of bees.

Further, the active ingredient of the medicament, feed or antidote comprises one or more of tea polyphenols or its homo-isomers. Can also contain other active ingredients besides tea polyphenols for improving bee immunity or antifungal ability.

Further, the medicament can also contain auxiliary materials allowed in the pharmaceutical field; the dosage form of the medicament can be any dosage form allowed in the pharmaceutical field.

Further, the feed may comprise tea polyphenols, and may also comprise tea polyphenols and various of their homologues; other active ingredients other than tea polyphenols (e.g., other active ingredients capable of enhancing the immunity or antiviral ability of bees) may also be included; in addition to the above active ingredients, the feed may also contain nutrients required by the bees, for example: pollen, lac Regis Apis, saccharide, yeast powder, inorganic salt, vitamins, water, etc.

In a sixth aspect, there is provided a product for use in the treatment or prophylaxis of bee poisoning, the active ingredient of the product comprising one or more of tea polyphenols or its homologues; the product is medicine, feed or feed additive.

The invention has the beneficial effects that:

1. the invention discovers for the first time that the tea polyphenol can obviously improve the survival rate of bees which eat the dinotefuran as a neonicotinoid insecticide by mistake and reduce the death rate of the bees. The experimental result shows that the death rate of bees eating the neonicotinoid insecticide by mistake after using tea polyphenol is greatly reduced, and the bees after detoxification are healthy; meanwhile, the food consumption of poisoned bees rises after tea polyphenol is fed; the learning and memory and crawling ability of bees are obviously up-regulated after the tea polyphenol is fed, the activities of detoxification enzyme, antioxidase and the like are reduced, the poisoning degree of apparent healthy bees can be improved by the intervention of the tea polyphenol, and the normal honey collection of bees is facilitated.

2. The invention provides a new function of tea polyphenol for bee neonicotinoid insecticide infection and provides a foundation for researching bee immune defense mechanism. The tea polyphenol can be applied to preparing medicines or feeds for preventing and controlling pesticides or improving the immunity of bees, provides a new method for preventing and controlling the pesticides, and has good market application prospect.

Drawings

FIG. 1 is a schematic diagram of the analysis of survival and mortality of tea polyphenols after intervention in healthy and infested bees;

FIG. 2 is a schematic diagram showing food consumption analysis after tea polyphenols intervene in the infested bees;

FIG. 3 is a schematic representation of the effect of tea polyphenol intervention on bee sucrose sensitivity and olfactory learning and memory behavior;

FIG. 4 is a schematic representation of the effect of tea polyphenol intervention on bee crawling ability;

FIG. 5 is a schematic representation of the effect of tea polyphenol intervention on bee enzyme activity.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1 intervention of healthy and toxic bees Using tea polyphenols

1. Sample specimen

Italian bees were from the institute of bee keeping in china (N39 female 59035.3300, E116 female 11059.7400). The bees tested were healthy and they were not exposed to the insecticide prior to the experiment.

2. Preparation of syrup

Preparation and feeding of syrups, reference is made to the standard for feeding Italian bee larvae in 2016 and adjustments are made on the basis of this standard (Karl Crailsheim, robert Brodschneider, pierrck Aupinel, diter Behrens, elke Genersch, jutta Vollmann & Ulrike Riessberger-Galle. Standard methods for artificial rearing of Apis mellifera larvae. Journal of Apicultural Research,2013,52 (1): 1-15.).

3. Preparation of tea polyphenols

Preparing 1g/l mother liquor by sterilized ultrapure water for later use. The test is carried out by using bee feed according to the requirements of each day of age, and the two concentrations of tea polyphenol (high concentration of tea polyphenol-100 mg/kg; low concentration of tea polyphenol-20 mg/kg) are obtained.

4. Preparation of neonicotinoid insecticide dinotefuran

Preparing 1mg/ml mother liquor with sterilized ultrapure water for later use. In the test, bee feed is used for dilution according to the requirements of each day of age, and the dinotefuran feeding concentration is taken as LC10 after acute exposure for 48 hours.

5. Sucrose sensitivity behavior test

On day 14 of rearing, 24 freshly-housed bees were randomly selected from each treatment group for the performance test of beak extension (PER). After the test bees are starved for 4 hours, the bees are fixed in the hollow plastic tube, so that the body is fixed, and the head is exposed outside the tube and can move freely. First, the susceptibility of bees to sucrose at different concentrations was tested: the tip cotton swabs filled with sucrose solutions with the concentration of 0.3%, 1%, 10%, and 30% are respectively and sequentially close to the antennae of bees for 3s, whether each bee actively emits the reflection of the extended beak is tested and recorded, each test concentration is spaced for 2 minutes, and the bees do not really eat sugar water in the test process. After the test is completed, 1 μl of 30% sugar water is administered to the bees.

6. Bee study and memory behavior test

After the susceptibility behavior test of bees to sucrose solutions with different concentrations, the bees are subjected to the study and memory behavior test, and as the normal susceptibility reaction of bees to sucrose solutions is the basis for testing the study and memory ability, bees which do not have the beak extension reflex behavior to any concentration of sucrose solution are eliminated in the study and memory behavior test. Then, the study and memory behavior test is carried out, firstly, the smell study is carried out, linalool is dipped by a cotton swab, the cotton swab contacts with the feeler of bees for 3s, and the beak-extension reaction is recorded. The test is divided into 2 phases, (1) a conditional stimulation phase (CS): the bees were kept in contact with linalool while in contact with bees 3s, while at the time of 3s the bees sucrose solution was given as a reward. (2) unconditional stimulation stage (US): keeping the main air flow and the odor stimulating air flow open for 3s, and observing whether bees actively generate beak extension reflection or not without using sucrose solution as a reward.

Learning ability test: and (3) after each bee completes 1 conditional stimulation, performing unconditional stimulation test, checking whether the bee actively generates beak extension reflex, and recording the number of times of using conditional stimulation when each bee generates the beak extension reflex for the first time.

Memory capability test: after the bees repeat the learning ability test for 3 times, unconditional stimulation test is carried out on the bees again to check whether the bees actively generate beak extension reflection or not and record. After each memory test, the immobilized bees were returned to the incubator and kept fed a small portion of sucrose solution every 1 hour to maintain the bees normal physiological response.

7. Bee crawling ability test

According to the phototaxis of bees, the self-made crawling device is utilized to measure the crawling ability of bees, namely, the crawling time from the dark lower part to the upper part is recorded under the guidance of a light source.

8. Variation of enzyme activity in bees

Each treatment randomly selects 12 bees, quickly freezing the bees with liquid nitrogen, quickly grinding the tissues into powder in a mortar, transferring the powder to a 1.5ml centrifuge tube, adding physiological saline, centrifuging the powder for 10min at the temperature of 2500 rpm in a frozen high-speed centrifuge at the temperature of 4 ℃, taking the supernatant, preparing crude enzyme liquid, and placing the crude enzyme liquid on ice to be tested. The protein content is measured by BCA protein concentration detection method, and the SOD, POD, ACHE, CAT, GSH, GR, NAD-MDH and LPO activities are measured according to the instruction book of Nanjing's built kit.

9. Test

Extracting a spleen of a capping seed to be taken out of a healthy bee colony with strong colony without obvious disease symptoms and pesticide pollution, placing the spleen into a queen bee oviposition limiting frame, culturing the spleen in a constant temperature incubator, collecting a sufficient number of young bees just taken out of the room after culturing the spleen in the incubator for 24 hours, randomly dividing the young bees into 7 groups, a ck control group, a cr solvent control group, dig is dinotefuran+high-concentration tea polyphenol treatment group, did is dinotefuran+low-concentration tea polyphenol treatment group, dis is dinotefuran+sucrose solution treatment group, stg is sucrose solution+high-concentration tea polyphenol treatment group, std is sucrose solution+low-concentration tea polyphenol treatment group, and repeating the steps for three times. Control group 14d was fed only sucrose solution; dig, did, dis dinotefuran was fed seven days before the three treatment groups, and 100mg/ml tea polyphenols, 20mg/ml tea polyphenols and 50% sugar water were fed after 7d treatment, respectively; seven days before three treatment groups of stg and std, a sucrose solution is fed, 100mg/ml of tea polyphenol and 20mg/ml of tea polyphenol are respectively fed after 7 days of treatment, a solvent control group is fed with the sucrose solution doped with DMSO, the feeding condition and the death rate of bees are observed and recorded after 14 days of exposure, and the learning and memory capacity and the crawling capacity of the bees are detected.

Example 2 analysis of survival and mortality of tea polyphenols after intervention in healthy and infested bees

As shown in figure 1, the survival rate of bees of a control group fed with sugar water and tea polyphenol can reach more than 80%, which indicates that the intervention of the tea polyphenol can not cause abnormal death of healthy bees and does not influence the growth and development of the healthy bees. The result shows that the intervention of tea polyphenol can prevent the infected bees from dying of illness, prevent the poisoned bees from collapsing, and play an important role in the recovery and reproduction of the bees.

The mortality statistics were carried out on each group of bees in example 1, and the measurement method of the bee mortality was as follows: the number of bees dead on the day of preculture and infection was not counted for analysis to exclude the effect of mechanical death of bees during the experiment, counting the number of bees dead at the timing of every day from the next day of infection, and removing dead bees, repeating the above steps until the 14-day-old period is over.

Daily bee mortality = number of deaths per day/number of bees survived per day x 100%;

overall process bee mortality = number of bee deaths/total number of samples x 100%.

The result shows that the daily death rate of the bees in the control group fed conventionally is very low, the daily death rate of the bees is unchanged after the intervention of the tea polyphenol, which indicates that the intervention of the tea polyphenol does not influence the growth and development of the bees, and the bee feeding control group can be used for the auxiliary feeding of the bees. The mortality rate of the bees after the treatment of the bee insecticide is greatly increased. The experimental bees survived only about 50% by 7 days of age. Compared with the treatment group, the death rate of bees at each day of age after the tea polyphenol is dried is greatly reduced, and the survival rate of bees at 7 days of age is up to more than 80 percent. The result shows that the intervention of tea polyphenol can greatly reduce the death rate of the infected bees, prevent the infected bees from collapsing, and play an important role in the recovery and reproduction of the bees.

Example 3 tea polyphenols intervention food consumption after contamination of bees

The bees in the treatment group and the control group of example 1 were examined, and as shown in FIG. 2, it was found that the food consumption of bees in the treatment group without intervention of tea polyphenols was greatly reduced. The result shows that toxic reaction is generated in the body of the infected bee after the pesticide is fed, and the food consumption of the bee is obviously increased after the intervention of the tea polyphenol, and the result shows that the toxicity of the pesticide to the bee is relieved after the tea polyphenol is fed, so that the appetite of the bee is improved.

Example 4 influence of tea polyphenol intervention on bee sucrose sensitivity and olfactory learning and memory behavior

To analyze the effect of tea polyphenol intervention on bee olfactory memory and learning memory, various groups of bees in example 1 were tested with different concentrations of sucrose. As shown in FIG. 3, after dinotefuran treatment, the beak extension rate of the dis treatment group was significantly lower than that of the dig and did treatment groups, and after tea polyphenol was added, the beak extension rate was significantly increased, almost in level with the ck group; meanwhile, the learning and memory rate of the ck group after tea polyphenol is added is slightly higher than that of the ck group. Similarly, as a result of decreased learning and memory in the dinotefuran-treated group, an increase occurred after addition of tea polyphenols. The result shows that the tea polyphenol has obvious effects of bacteriostasis, antivirus and the like, and can effectively remove free radicals and toxins in the body; protecting cells from free radical attack, inhibiting proliferation of pathogenic bacteria, and has good anti-stress effect, and relieving production performance decrease caused by stress.

Example 5 Effect of tea polyphenol intervention on bee crawling ability

As shown in fig. 4, the crawling ability of bees after adding tea polyphenol is significantly higher than that of dinotefuran-treated group, and the crawling time after intervention with tea polyphenol is slightly lower than that of control group. The result shows that the tea polyphenol can improve the damage after dinotefuran is fed and reduce the crawling time of bees.

Example 6 Effect of tea polyphenol intervention on bee enzyme Activity

As shown in fig. 5, after chronic exposure for 14d, it can be seen from the figure that the dinotefuran-fed group almost significantly increased the individual enzyme activities, whereas the individual enzyme activities significantly decreased after dry tea polyphenol. The result shows that the enzyme activities of the antioxidant enzyme, the detoxification enzyme and the like in the bee body are obviously reduced after the tea polyphenol is dried, which indicates that the tea polyphenol plays a certain role in resisting oxidation and detoxification in the bee body.

The invention takes bees treated by insecticide as research objects, utilizes the sensitivity degree and learning condition of bees to sugar water with different concentrations, carries out memory detection on the expression condition of tea polyphenol in the stages of insecticide poisoning and healthy bees, and increases the food consumption after feeding the tea polyphenol, thereby defining the detoxification capability of the tea polyphenol; the intervention of tea polyphenol strongly resists the invasion of the insecticide to bees, reduces the death rate of the bees, forms a rapid and effective defense mechanism, and is used for rapidly killing or eliminating exogenous pathogenic microorganisms and protecting the bees from normally picking up honey.

In conclusion, the tea polyphenol can improve the immunity of organisms, improve the digestion and absorption rate and promote the growth of animals. On the other hand, the tea polyphenol has obvious effects of antioxidation, bacteriostasis, antivirus and the like, and can effectively remove free radicals and toxins in the body; protecting cells from free radical attack, promoting metabolism, inhibiting proliferation of pathogenic bacteria, and relieving production performance decrease caused by stress. Therefore, can be used for preventing and treating bee insecticide poisoning.

According to the effect of tea polyphenols on alleviating bee poisoning, substances containing phenolic compounds and capable of being metabolized in vivo to produce phenols or other phenolic derivatives should have the same effect. The derivative containing the phenolic compound structure comprises various compounds such as phenols, flavones, isoflavones, tannins and the like.

The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

Use of tea polyphenols at a low concentration of 1.20mg/ml as sole active ingredient for the preparation of an antidote for the neonicotinoid insecticide dinotefuran.