TWI612899B - Camellia seed extract and biopesticides with the same - Google Patents

Abstract

The invention relates to an extract of camellia oleifera meal and biological pesticides used therein, wherein the components of the extract of camellia oleifera meal include: triterpenoid saponin compounds 0.5 to 99%, flavonoid compounds 0.5 to 40%, and further containing phenols Compound 0-20%. The biological pesticide contains the camellia meal extract, which can be used for controlling mollusks, nematodes or inhibiting the growth of plant disease microorganisms.

Description

Camellia oleifera meal extract and biological pesticide made therefrom

The invention relates to a plant extract and a biological pesticide made therefrom, in particular to an extract obtained by extracting camellia oleifera meal, and a biological pesticide made therefrom for controlling mollusks, nematodes or inhibiting plant diseases. Microbial growth, etc.

In recent years, environmental awareness has risen. Since natural saponin can be rapidly decomposed by microorganisms and does not impact the environment, the use of saponin derived from plants has been re-emphasized. For example, the regeneration of camellia meal (also known as bitter tea meal). Camellia oleifera meal is derived from the oily residue of bitter tea tree seeds. Because it is rich in tea saponin, it can not only be used as a natural cleanser, but also can damage the snail's mucosa and cause it to die. Fertilization and Fushou snail control.

The practical application of tea saponin in agriculture is still mostly based on direct application of camellia oleifera meal or its extracts and other related products. For example, during rice transplanting, the untreated camellia meal was sprayed in the paddy field, so that the tea saponin was released from the tea saponin in the water, which caused the death of Fushou snail exposed to the tea saponin, which effectively inhibited the growth of Fushou snail. The amount is high and the volume is large, which causes a burden on field application. In addition, the camellia meal can be immersed in water to dissolve the tea saponin in water. After simple filtering, it is a crude extract, which is used as a raw material and then processed to produce various extracts. Due to the large volume of the final product of the extraction liquid, it is difficult to store and transport. If the water in the extract is to be evaporated to separate the extract, it is a high energy-consuming process, which will inevitably increase the manufacturing cost.

Taiwan Patent No. I379896 provides a natural saponin preparation method, which uses a silicon-containing solid phase medium as the adsorbent of natural saponin. Therefore, the finished product is a powdered natural saponin, thereby reducing the volume and eliminating the need for High energy consumption.

The invention provides a camellia meal extract which is different from the traditional saponin preparation method, which has high biologically active ingredients and simple ingredients.

Specifically, the camellia meal extract produced by the special tea saponin extraction and purification process of the present invention is characterized by being composed of the following weight percentage components: triterpenoid saponin compounds 0.5 to 99%; flavonoid compounds 0.5 to 40%; and further containing 0 to 20% of phenolic compounds. The flavonoids include flavonols.

The camellia meal extract of the present invention can be further produced into a biological pesticide with a specific composition ratio for controlling molluscs such as flat snails, African snails and snails, and root nodule nematodes, and can also inhibit plant disease microorganisms such as penicillium, green The growth of microorganisms such as mold, Rhizoctonia solani, Fusarium oxysporum, and anthracnose.

Specifically, the biological pesticide of the present invention can be targeted at different dosage forms including baits, water-soluble granules or granules, and water-soluble powders, solutions, emulsions or microemulsions. When a biological pesticide is made into a bait, its composition further includes an attractant, a preservative, a filler and a binding agent; and when the biological pesticide is made into a water-soluble granule, its composition further includes a dispersant , A binder, a disintegrant and a carrier.

The camellia meal extract of the present invention is prepared by a tea saponin extraction and purification process (first picture), which has high biologically active ingredients. The active ingredients mainly include triterpenoid saponins and similar compounds. Flavonoids can be used to control molluscs and rhizobial nematodes such as flat snails, large African snails and snails, and can also inhibit penicillium, green mold, Rhizoctonia solani, Fusarium oxysporum, and anthracnose Wait for the growth of microorganisms.

Referring to the first figure, the tea saponin extraction and purification process roughly includes five stages including pre-treatment 1, degreasing 2, tea saponin extraction 3, tea saponin purification 4, and post-treatment 5, and the details are as follows:

First, in the pre-treatment stage 1, the camellia meal sample (oil camellia meal cake) is first pulverized with a grinder (step 11) and sieved with a sieve of ≧ 60 mesh to increase its contact surface area with the solvent, and Weigh the sifted camellia meal powder.

Generally speaking, camellia oleifera meal is a residue of the tea seed derived from bitter tea tree after being pressed. Although it is rich in tea saponin, in fact, even if there is still a lot of oil and fat in the camellia meal (cake) after the oil is pressed, Prior to the extraction of tea saponin, it is preferred to perform a degreasing operation 2 on the camellia meal. At this stage, the sieved powder is added to a fat-soluble organic solvent, such as n-hexane (material-liquid ratio 1: 4), and stirred and mixed uniformly to dissolve the fat to extract the fat (step 21). This step was performed by ultrasonic extraction at room temperature for 60 minutes. It is then filtered (step 22) to remove the solvent n-hexane. After repeating the process several times, the degreased camellia meal powder is air-dried (step 23).

Continue to the stage 3 of tea saponin extraction. First, add the dried camellia meal powder to a mixed solvent of methanol and acetone, and stir and mix well to dissolve the tea saponin in the mixed solvent to extract tea saponin. (Step 31). In this step, extraction is performed at room temperature for 60 minutes with ultrasonic vibration to improve the extraction efficiency, followed by filtering (step 32) to filter out impurities and collect the filtrate, which is the tea saponin extract. This is repeated many times. Next, the filtered tea saponin extract is concentrated under reduced pressure (step 33) to remove the solvent and obtain a high-concentration tea saponin extract. Subsequently, a precipitating agent is used for the precipitation, and the precipitating agent is a substance such as ketones or ethers. Since tea saponin is insoluble in precipitants such as ketones and ethers, it will precipitate due to the addition of a precipitant, so after pouring the supernatant, a precipitated crude tea saponin is obtained (step 34).

Then enter the stage 4 of tea saponin purification. In this stage, the tea saponin precipitate is re-dissolved with a high temperature alcohol-mixed solvent (step 41), and the above-mentioned precipitant is added to precipitate (step 42). This cycle is repeated many times to purify tea saponin.

Finally, in the stage 5 of post-treatment, the purified tea saponin precipitate is washed with an appropriate amount of a ketone solvent (step 51), and then dried in an oven (step 52) to obtain an oil-tea camellia meal extract, and record its weight. The yield is about 5 ~ 25%.

Today, the ultra-high performance liquid chromatography tandem mass spectrometer (UPLC / ESI / MS / MS) with electrospray method was used to identify and analyze the extracts of camellia oleifera meal, and the total ion chromatogram shown in the second figure was obtained. . From this map, it can be seen that the constituents of the camellia meal extract mainly include three groups A, B, and C. Among them, the peak of group A appears at a residence time of 0.6-1.0 minutes, and the structure analysis of the primary mass spectrum, its secondary mass spectrum, and triple mass spectrum can be used to estimate that such compounds are phenolic compounds. The peak of group B is at a residence time of 4.5-6.0 minutes. Through the analysis of the structure of the primary mass spectrum, the secondary mass spectrum, and the triple mass spectrum, it can be estimated that these compounds are flavonoids, especially flavonol glycosides. Among them, the peak of group C is at a retention time of 10.6 to 16.7 minutes. Through the analysis of the structure of the primary mass spectrum, the secondary mass spectrum, and the triple mass spectrum, it can be estimated that such compounds are triterpenoid saponin compounds.

After several tea saponin extraction and purification experiments, it was found that the composition of the camellia meal extract produced by the present invention is very simple, and is mainly composed of the following weight percentages: triterpenoid saponin compounds 0.5 to 99% ; Flavonoids from 0.5 to 40%; and further containing phenolic compounds from 0 to 20%. Among them, triterpenoid saponin compounds and flavonoids are the main biologically active components. In addition, the molecular weights of various components in the triterpenoid saponin compound fall within a specific range, as if through screening, it is shown that the components of the triterpenoid saponin compound are simple and special.

After the qualitative and quantitative analysis of the aforementioned mass spectrometer, the present invention further makes the Camellia oleifera meal extract into a biological pesticide with a specific content ratio, and according to the different needs of the control object or the use method, it is made into a corresponding dosage form, such as a bait. , Granules, solutions, emulsions, microemulsions, water-soluble granules or water-soluble powders. Among them, the control targets mainly include flat snails, large African snails, and snails such as snails, rhizobial nematodes, and Penicillium, green mold, Rhizoctonia solani, Fusarium oxysporum, and anthracnose.

The third picture shows the process of further making the camellia meal extract into a biological pesticide. First, the camellia oleifera meal extract and other materials are used as raw materials, which are kneaded, extruded and granulated, and then dried, granulated, and other steps to form the biological pesticide containing the camellia oleifera meal extract. Wherein, when the biological pesticide needs to be made into a bait dosage form, the raw material includes an attractant, a preservative, a filler and a binding agent in addition to the camellia meal extract; and when the biological pesticide needs to be made into a water solvent In the form of granules, the raw materials include a dispersant, a binder, a disintegrant, and a carrier in addition to the camellia meal extract.

The biological pesticide of the present invention has been tested for activity, and it has been confirmed that it is effective in controlling molluscs such as flat snails, African snails, and snails, controlling root nodules and inhibiting penicillium, green mold, Rhizoctonia solani, Fusarium oxysporum and anthracnose All aspects such as the growth of plant disease microorganisms have excellent control effects, as shown in Figures 4 to 15 (see attachment).

Activity test (1): African snail

Refer to the statistical table in the fourth figure, take five pots for five African snails, and sprinkle about 2 grams of bait containing 7.9% camellia meal extract in each pot. After six days of observation, statistics The life state found that the corrected mortality rate was 60%, and the corrected mortality rate was 75% after ten days. Therefore, the bait containing the camellia meal extract of the present invention can indeed kill African snails.

Activity test (2): Flat snail

Refer to the statistical table in the fifth figure. Take three pots for 50 flat snails. The first pot is not applied with any pesticide (control group), and the second pot is sprinkled with 7.9% camellia meal. The bait of the extract is about 2 grams (test group), and the third pot is sprinkled with about 2 grams of the conventional 6% polyacetaldehyde pesticide. After observing for 164 hours, the life state of the three pots was found. The mortality of flat snails was 30%, 84%, and 92%, so it can be known that the bait containing the camellia meal extract of the present invention has a control effect on flat snails comparable to the conventional polyacetaldehyde pesticide.

Activity test (3): flat snails on cabbage plants

Refer to the statistical chart in Figure 6. Dilute the water-soluble granules containing 34.62% camellia meal extract to 0.46 mg / mL, 0.68 mg / mL, and 0.88 mg / mL, and apply them to cabbage plants. Observe ten Two days later, the life status of flat snails was found to have a LC50 of 0.88 mg / mL in 12 days. In addition, during the 12-day period, it was observed that the flat snail only walked and climbed on the plant before death, and did not eat the plant, making the surface of the plant very complete. It is speculated that the activity of the camellia meal extract of the present invention The ingredients can also inhibit the taste receptors of the flat snail, making it unable to recognize food normally, and produce antifeedant effect, and the higher the concentration, the more obvious the effect.

Activity test (4): Flat snail on dragon fruit plant

Refer to the statistical chart in Figure 7. Dilute the water-soluble granules containing 34.62% camellia meal extract to 0.89 mg / mL, 1.79 mg / mL, and 2.61 mg / mL to apply the dilution to the dragon fruit plant. Observe Twelve days later, the life status of the flat snail was calculated to find that the LC50 was 0.89 mg / mL. Furthermore, during the 12-day period, it was observed that the flat snail only walked and climbed on the plant before death, and did not eat the plant, so the plant was complete. Once again, the active ingredients of the camellia meal extract were verified in the dragon fruit. Plants or cabbage plants are antifeedant to flat snails.

Activity test (5): Fushou

Referring to the statistical table in Figure 8, the water-soluble powder containing 86.51% camellia meal extract was diluted to a dilution of 70 μg / mL and the water-soluble granules containing 34.62% camellia meal extract were diluted to 70 μg / mL. At the same time, the snails were tested at the same time. It was found that after 72 hours, all snails died, and the mortality rate was 100%. In other words, whether it is water-soluble powder or water-soluble granules, the snails can be lethal to death.

Activity test (6): Nodular nematode

Referring to the statistical chart in Figure 9, the nodule nematodes were tested by diluting camellia oleifera extract to 0.25 mg / mL, 1.00 mg / mL, 3.98 mg / mL and 7.97 mg / mL, and found that the higher the concentration , The more lethal effect on root nodule nematodes, especially the dilution at a concentration of 7.97 mg / mL has a lethal rate as high as 100%, confirming that it can effectively inhibit the growth of nodular nematodes.

Activity test (7): Rhizobial nematodes on plants

Referring to the statistical graph in Fig. 10, a 1 g water soluble granule containing 34.62% camellia oleifera meal extract was used to perform a plant growth test on the nematode nematode (experimental group), and it was found that compared with the non-applied control group, not only the nodule index Lower, only about 40% of the root system has root nodule nematodes, and the number of nematodes in the unit soil is significantly smaller, so it can effectively inhibit the growth of nodular nematodes.

Activity test (8): Penicillum italicum

Referring to the data table in Figure 11, the penicillium spp. Was tested after diluting the water-soluble powder of camellia oleifera extract with an appropriate concentration of diluent. The average growth radius of the control group without application was 2.52 cm. The average growth radius of the test group was only 0.2 cm. Looking further, the average growth radius (0.2 cm) of this test group minus the mycelial mass radius (0.2 cm) is the net growth radius equal to zero. In other words, the inhibition rate is 100%, which proves that it has a good effect on penicillium Excellent antibacterial effect.

Activity test (9): Green mold fungus (penicillum digitatum)

Refer to the data table in Figure 12. After testing the green mold fungus after diluting the water-soluble powder of camellia oleifera meal extract with an appropriate concentration, it was found that the average growth radius of the control group without application was 2.72 cm. With the higher concentration of the test group, the smaller the growth diameter, the better the bacteriostatic effect. The average growth radius of the test group-4 (0.35 cm) after deducting the mycelial mass radius (0.2 cm) was the net result. The growth radius is even as small as 0.15 cm, indicating that the inhibition rate can be as high as 94.04%, confirming that it has an excellent antibacterial effect on green mold.

Activity test (10): Rhizoctonia solani

Refer to the data table in the thirteenth figure. After testing the Rhizoctonia solani with the water-soluble powder of Camellia oleifera meal extract to the appropriate concentration, the average growth radius of the control group was 3.23 cm. However, As the test group with the application increased, the smaller the growth diameter, the better the bacteriostatic effect. The average growth radius of the test group-4 (0.78 cm) was subtracted from the mycelial mass radius (0.2 cm). The net growth radius is equal to 0.58 cm, indicating that the inhibition rate can reach 80.77%, confirming that it has a good antibacterial effect on Rhizoctonia solani.

Activity test (11): Fusarium oxysporum

Referring to the data table in Figure 14, after testing the Fusarium oxysporum after diluting the water-soluble powder of camellia oleifera meal extract with an appropriate concentration, it was found that the average growth radius of the control group without application was 3.68 cm. However, With the increase in concentration, the growth diameter of the test group decreased, indicating a better antibacterial effect. The average growth radius of the test group-3 (2.42 cm) was subtracted from the mycelial block radius (0.2 cm). The net growth radius is equal to 2.22 cm, which indicates that the inhibition rate can reach 36.36%, confirming that it has a moderate antibacterial effect on Fusarium oxysporum.

Activity test (12): Colletotrichum gloeosporioides

Referring to the data table in Figure 15, after testing the anthracnose bacteria with a water-soluble powder of Camellia oleifera meal extract diluted to an appropriate concentration, it was found that the average growth radius of the control group without application was 4.25 cm. As the concentration of the test group increased, the growth diameter became smaller, indicating a better bacteriostatic effect. The average growth radius of the test group-4 (2.08 cm) was subtracted from the net growth of the mycelium mass (0.2 cm). The radius is equal to 1.88 cm, which indicates that the inhibition rate can reach 53.50%, which confirms that it has a good antibacterial effect on anthracnose.

As described above, the special tea saponin extraction and purification process of the present invention can obtain a purified camellia oleifera meal extract, the composition of which has high biological activity and is simple. In addition, the composition is qualitative and quantitative with ultra-high performance liquid chromatography-tandem mass spectrometry. Analysis shows that the biological pesticide produced by the camellia meal extract of the present invention can be identified and quantitatively controlled, and can effectively control molluscs such as flat snails, African snails and snails, control root nodules and inhibit penicillium, green mold, Growth of plant diseases such as Rhizoctonia solani, Fusarium oxysporum and anthracnose.

In any case, anyone can get enough teaching from the description of the above examples, and understand that the content of the present invention is indeed different from the prior art, and has industrial applicability and is sufficiently progressive. It is true that the present invention has met the patent requirements, and the application was filed according to law.

1‧‧‧ pre-processing

11‧‧‧ sample crushing

2‧‧‧ degreasing

21‧‧‧Extract Fat

22‧‧‧Filter

23‧‧‧ dry

3‧‧‧Tea Saponin Extract

31‧‧‧Tea saponin extraction

32‧‧‧Filter

33‧‧‧Concentrated under reduced pressure

34‧‧‧ Tea saponin precipitation

4‧‧‧ Purification of tea saponin

41‧‧‧Tea saponin redissolved

42‧‧‧ Tea saponin precipitation

5‧‧‧ post-processing

51‧‧‧Cleaning

52‧‧‧ drying

The first figure is a manufacturing flow chart of tea saponin extraction and purification of the present invention. The second figure is the identification map of the camellia meal extract of the present invention. The third figure is a manufacturing flow chart of the biological pesticide of the present invention. The fourth figure is a data table of the lethality of the biological pesticide of the present invention to the African snail. The fifth figure is a data table of the lethality of the biopesticide of the present invention to the snail. The sixth figure is a data chart of the lethality of the biological pesticide of the present invention diluted to different concentrations on flat snails on cabbage plants. The seventh figure is a data chart of the fatality rate of the flat snail on the dragon fruit plant when the biological pesticide of the present invention is diluted to different concentrations. The eighth figure is a data table of the lethality of the biological pesticide of the present invention diluted to different concentrations on the snail. The ninth figure is a data chart of the lethality of the nodule nematodes diluted by the biological pesticide of the present invention to different concentrations. The tenth figure is a data chart of the inhibitory effect of the biopesticide of the present invention on nodule nematodes on plants. The eleventh figure is a data table of the bacteriostatic effect of the biological pesticide of the present invention on Penicillium spp. The twelfth figure is a data table of the bacteriostatic effect of the biological pesticide of the present invention on green mold. The thirteenth figure is a data table of the antibacterial effect of the biological pesticide of the present invention on Rhizoctonia solani. The fourteenth figure is a data table of the bacteriostatic effect of the biological pesticide of the present invention on Fusarium oxysporum. The fifteenth figure is a data table of the bacteriostatic effect of the biological pesticide of the present invention on anthracnose.

1‧‧‧ pre-processing

11‧‧‧ sample crushing

2‧‧‧ degreasing

21‧‧‧Extract Fat

22‧‧‧Filter

23‧‧‧ dry

3‧‧‧Tea Saponin Extract

31‧‧‧Tea saponin extraction

32‧‧‧Filter

33‧‧‧Concentrated under reduced pressure

34‧‧‧ Tea saponin precipitation

4‧‧‧ Purification of tea saponin

41‧‧‧Tea saponin redissolved

42‧‧‧ Tea saponin precipitation

5‧‧‧ post-processing

51‧‧‧Cleaning

52‧‧‧ drying

Claims (14)

An extract of camellia oleifera meal contains triterpenoid saponins, which accounts for 0.5 to 99% by weight of the extract of camellia oleifera meal; and a flavonoid compound, which accounts for the weight percentage of the extract of camellia oleifera It is 0.5 ~ 40%. The oil-tea camellia meal extract as described in item 1 of the scope of the patent application, further comprising a phenolic compound, which accounts for 0% to 20% by weight of the oil-tea camellia meal extract. The extract of camellia oleifera meal according to item 1 of the scope of patent application, wherein the flavonoids include flavonols. A biological pesticide, the composition of which includes the camellia meal extract as described in item 1 or 2 of the scope of the patent application, and its control object is mollusk, nematode or plant disease microorganism. According to the biological pesticide described in item 4 of the application, the dosage form is any one of bait, water-soluble granule, granule, water-soluble powder, solution, emulsion and microemulsion. The biological pesticide described in item 5 of the scope of patent application is a bait for controlling flat snails, large African snails, slugs or snails. According to the biological pesticide described in item 6 of the patent application scope, the composition further includes an attractant, a preservative, a filler and a binding agent. The biological pesticide described in item 5 of the scope of application for a patent is a water-soluble granule for controlling flat snails, large African snails, slugs, snails, nematodes or fungi. According to the biological pesticide described in item 8 of the scope of the patent application, its components further include a dispersant, a disintegrant, a binder and a carrier. The biological pesticide described in item 5 of the scope of application for a patent is a water-soluble powder for controlling Fushou snail, flat snail, African snail, tadpole, snail, nematode or fungus. According to the biological pesticide described in item 10 of the patent application scope, the composition further includes a preservative and a filler. A biological pesticide, which comprises the camellia oleifera meal extract as described in item 1 or 2 of the scope of patent application, and its control object is a root nodule nematode. A biological pesticide, which comprises the camellia meal extract as described in item 1 or 2 of the scope of patent application, for inhibiting the growth of plant disease microorganisms. The biological pesticide according to item 13 of the application, wherein the plant disease microorganism is selected from the group consisting of penicillium, green mold, Colletotrichum gloeosporioides , Fusarium oxysporum, and Rhizoctonia solani Group of bacteria (Rhizoctonia solani).