CN113057265A - A kind of juvenile salamander feed additive and preparation method thereof - Google Patents
A kind of juvenile salamander feed additive and preparation method thereof Download PDFInfo
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- CN113057265A CN113057265A CN202110406770.6A CN202110406770A CN113057265A CN 113057265 A CN113057265 A CN 113057265A CN 202110406770 A CN202110406770 A CN 202110406770A CN 113057265 A CN113057265 A CN 113057265A
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- China
- Prior art keywords
- feed additive
- coilia ectenes
- feed
- gambogic acid
- acid derivative
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- 238000002360 preparation method Methods 0.000 title claims abstract description 17
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- GEZHEQNLKAOMCA-UHFFFAOYSA-N epiisogambogic acid Natural products O1C2(C(C3=O)(CC=C(C)C(O)=O)OC4(C)C)C4CC3C=C2C(=O)C2=C1C(CC=C(C)C)=C1OC(CCC=C(C)C)(C)C=CC1=C2O GEZHEQNLKAOMCA-UHFFFAOYSA-N 0.000 claims description 15
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Images
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/116—Heterocyclic compounds
- A23K20/137—Heterocyclic compounds containing two hetero atoms, of which at least one is nitrogen
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/174—Vitamins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
- A23K20/30—Oligoelements
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/12—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains three hetero rings
- C07D493/18—Bridged systems
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Animal Husbandry (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physiology (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- Inorganic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Insects & Arthropods (AREA)
- Marine Sciences & Fisheries (AREA)
- Birds (AREA)
- Fodder In General (AREA)
- Feed For Specific Animals (AREA)
Abstract
The invention discloses a feed additive for coilia ectenes juvenile fish and a preparation method thereof, and relates to the technical field of aquatic fish feeds. The feed additive comprises, by weight, 2-4 parts of iris tonkinensis root extract, 1-4 parts of beta-L-aspartyl-L-lysine, 0-4 parts of gambogic acid derivative, 0.5-2 parts of oligosaccharide, 0.5-0.9 part of selenium methionine, 0.3-0.7 part of vitamin C phosphate and 0.01-0.04 part of digestive enzyme. The feed additive prepared by the invention can effectively improve the survival rate of the coilia ectenes juvenile fish, promote the digestion and absorption capacity of the coilia ectenes juvenile fish to the feed and enhance the fullness of the coilia ectenes; and the expression of stress related genes can be obviously influenced, and the oxidative stress caused by the transport stress of the coilia ectenes can be effectively relieved.
Description
Technical Field
The invention belongs to the technical field of aquatic fish feeds, and particularly relates to a feed additive for coilia ectenes juvenile fish and a preparation method thereof.
Background
Yangtze river Coilia ectenes (Coilia ectenes Jordan et Seale), commonly known as swordfish, hairtail, wild hairtail, Coilia ectenes, etc., and Yangtze river hilsa herring (Tenualosa reevesii) and globefish (Takifugu) are also known as "Yangtze river Sanxian". The coilia ectenes belong to migratory fishes, live in the sea at ordinary times, enter the river from the sea in 2-4 months every year, and perform reproductive migration on the river. Spawning groups enter lakes and tributaries along the Yangtze river or spawning activities are carried out in the main stream of the Yangtze river. Due to the aggravation of Yangtze river pollution and the excessive fishing and fishing, the yield of Yangtze river hairtail is reduced year by year. And the miniaturization of the Yangtze river coilia ectenes population and individuals is serious. Coilia ectenes resources are endangered and died, and coilia ectenes germplasm resource protection is not easy to be developed. Coilia ectenes cannot form dominant species in Yangtze river, and the price of commercial coilia ectenes in Yangtze river is continuously increased. Therefore, the breakthrough and the popularization of the artificial breeding technology of the coilia ectenes promote the industrialized culture production of the coilia ectenes, can promote the adjustment of the fishery industry structure, and promote the fishery efficiency improvement and the fisherman income increase.
In the process of cultivating the fries of the coilia ectenes, the screening of the suitable bait is an important work, the types of the bait are continuously changed at different periods of the individual development of the fish, and the survival rate of the fries of the coilia ectenes can be improved and the growth speed can be accelerated by feeding the suitable bait. Under natural conditions, the coilia ectenes has wide feeding habits and has both selectivity and randomness, mainly takes cladocera, copepods, small fishes, shrimps and the like of zooplankton in water as food, and is limited in large-scale intensive culture due to the difficulty in supplying fresh live baits during artificial culture. With the development of artificial culture of the coilia ectenes, if the bait is still mainly natural bait, the large-scale culture of the coilia ectenes is seriously restricted. Therefore, the artificial domestication of the coilia ectenes and the development of palatable artificial feed can not only greatly reduce the culture cost and improve the culture benefit, but also enlarge the culture scale and enhance the feasibility of the coilia ectenes culture industrialization.
Disclosure of Invention
The feed additive can effectively improve the survival rate of the coilia ectenes juvenile fish, promote the digestion and absorption capacity of the coilia ectenes juvenile fish on the feed and enhance the fullness of the coilia ectenes; and the expression of stress related genes can be obviously influenced, and the oxidative stress caused by the transport stress of the coilia ectenes can be effectively relieved.
The technical scheme adopted by the invention for realizing the purpose is as follows:
the use of a gambogic acid derivative in the preparation of a feed additive, the gambogic acid derivative having the structural formula shown below:
the gambogic acid derivative obtained by modifying the gambogic acid structure by adopting 4-aminobenzoic acid-1H-benzimidazole-2-methyl ester can obviously change the antibacterial property of the gambogic acid derivative and generate effective inhibition effect on escherichia coli, and the inhibition effect on gram-positive bacteria staphylococcus aureus by grafting the 4-aminobenzoic acid-1H-benzimidazole-2-methyl ester is obviously enhanced. The gambogic acid derivative is applied to the feed additive, and plays a role in enhancing the growth of coilia ectenes juvenile fish. The survival rate of the coilia ectenes juvenile fish can be effectively improved, and the fullness of the coilia ectenes juvenile fish can be increased; and has certain influence on the expression of UI and GR genes, and the fish body loss caused by stress reaction in the transportation process is improved.
Preferably, the gambogic acid derivative is prepared by grafting 4-aminobenzoic acid-1H-benzimidazole-2-methyl ester onto gambogic acid through acylation reaction.
Further, the preparation method of the gambogic acid derivative specifically comprises the following steps:
adding DMF into gambogic acid, EDCI, DMAP and HOBT under the protection of nitrogen, and stirring at room temperature: adding 4-aminobenzoic acid-1H-benzimidazole-2-methyl ester diluted by DMF (dimethyl formamide) after 25-30 min, stirring at room temperature for reacting overnight, pouring the reaction liquid into water, and sequentially extracting with ethyl acetate for 2-3 times, wherein each time is 12-15 mL; combining the organic phases, drying the organic phases by using anhydrous magnesium sulfate, filtering the magnesium sulfate, concentrating the ethyl acetate phase at 40-45 ℃ under reduced pressure, and evaporating the ethyl acetate phase to dryness to obtain a yellow crude product; the product was separated by column chromatography (ethyl acetate: dichloromethane ═ 1: 11) to give a gambogic acid derivative.
Preferably, the molar ratio of gambogic acid, 4-aminobenzoic acid-1H-benzimidazole-2-methyl ester, EDCI, DMAP and HOBT is 1: 1.9-2.2: 2-2.1: 2-2.2: 1 to 1.2; the solid-liquid ratio of the gambogic acid to the DMF is 20-24 mg: 1 mL.
It is a further object of the present invention to provide the use of a gambogic acid derivative for enhancing the survival and fullness of coilia ectenes.
A feed additive comprising: the gambogic acid derivative described above.
Preferably, the feed additive comprises the raw material components of, by weight, 2-4 parts of iris soloensis root extract, 1-4 parts of beta-L-aspartyl-L-lysine, 0-4 parts of gambogic acid derivative, 0.5-2 parts of oligosaccharide, 0.5-0.9 part of selenium methionine, 0.3-0.7 part of vitamin C phosphate and 0.01-0.04 part of digestive enzyme. The feed additive contains the iris tectorum root extract and/or the gambogic acid derivative, and is added into feed to feed coilia ectenes juvenile fishes, so that the lipase content in the body of the coilia ectenes juvenile fishes is obviously improved, the digestion and absorption capacity is obviously enhanced, the feed conversion rate can be effectively promoted, the fat synthesis is greater than the decomposition, the fat deposition exists in the body, the specific growth rate is obviously increased, and the fullness of the coilia ectenes is further effectively improved; the survival rate of the coilia ectenes juvenile fish can be effectively improved; the two are added simultaneously, and the compound usage has better enhancing effect on the fullness and survival rate of the coilia ectenes. In addition, the gambogic acid derivative has a promoting effect on the expression of UI and GR genes, influences downstream metabolic activity to a certain extent and improves the change of biochemical indexes related to oxidative stress.
Preferably, all the raw material components are powder, and the feed additive is obtained by uniformly mixing and stirring the powder components.
Preferably, the irigenin A content in the extract of Iris pallida is not less than 33.2%, and the irigenin A content is not less than 29.3%.
Preferably, the digestive enzymes comprise one or more of proteases and cellulases.
Preferably, the feed additive is added into the feed according to the weight percentage of 0.2-0.5%.
Furthermore, the raw material components of the feed additive also comprise 0.02-0.06 part by weight of (5E) -5- (4-ethoxy benzylidene) -2-mercapto-1, 3-thiazole-4 (5H) -ketone.
The addition of (5E) -5- (4-ethoxy benzylidene) -2-mercapto-1, 3-thiazole-4 (5H) -ketone in the feed additive can effectively influence the expression of stress related genes such as CRH, UI, POMC and GR, reduce the occurrence probability of inducing oxidative stress reaction, further effectively relieve the oxidative stress caused by the transportation stress of the coilia ectenes, and avoid the damage of tissues such as liver and the like to a certain extent to cause the irreversible physiological change of organisms; and the improving effect on the survival rate of the coilia ectenes juvenile fish is obviously enhanced.
Still another object of the present invention is to provide use of the feed additive for preparing a feed for increasing the survival rate and the feed intake rate of coilia ectenes larvae.
Compared with the prior art, the invention has the following beneficial effects:
due to the existence of the iris tectorum root extract and/or the gambogic acid derivative in the feed additive, the lipase in the coilia ectenes juvenile fish is remarkably improved, the digestion and absorption capacity is remarkably enhanced, the feed conversion rate can be effectively promoted, fat deposition exists in the coilia ectenes, and the fullness of the coilia ectenes is further effectively improved; the survival rate of the coilia ectenes juvenile fish can be remarkably improved; the two are added simultaneously, the compound usage has better effect of enhancing the fullness and survival rate of the coilia ectenes, and the addition of the gambogic acid derivative has the effect of promoting the expression of UI and GR genes, influences the downstream metabolic activity to a certain extent and improves the change of biochemical indexes related to oxidative stress. In addition, the feed additive is added with (5E) -5- (4-ethoxy benzylidene) -2-mercapto-1, 3-thiazole-4 (5H) -ketone, which can effectively influence the expression of stress related genes such as CRH, UI, POMC and GR, reduce the occurrence probability of inducing oxidative stress reaction, further effectively relieve the oxidative stress caused by the transportation stress of the coilia nasus, and avoid the damage of tissues such as liver and the like to a certain extent to cause the irreversible physiological change of organisms; and the survival rate of the coilia ectenes juvenile fish can be further improved.
Therefore, the feed additive for the coilia ectenes juvenile fish and the preparation method thereof can effectively improve the survival rate of the coilia ectenes juvenile fish, promote the digestion and absorption capacity of the coilia ectenes juvenile fish on the feed and enhance the fullness of the coilia ectenes; and the expression of stress related genes can be obviously influenced, and the oxidative stress caused by the transport stress of the coilia ectenes can be effectively relieved.
Drawings
FIG. 1 shows the results of the measurement of the relative expression level of mRNA in test example 3 of the present invention.
Detailed Description
The technical solution of the present invention is further described in detail below with reference to the following detailed description and the accompanying drawings:
the extract of iris pallida used in the examples of the present invention was purchased from seoul biotechnology limited.
Preparation of gambogic acid derivatives:
taking gambogic acid, EDCI, DMAP and HOBT (the molar ratio of the gambogic acid to the EDCI, the DMAP to the HOBT is 1: 2: 2.1: 1.1), adding DMF (the solid-to-liquid ratio of the gambogic acid to the total DMF is 23.4 mg: 1mL) under the protection of nitrogen, stirring at room temperature: adding 4-aminobenzoic acid-1H-benzimidazole-2-methyl ester (the molar ratio of the 4-aminobenzoic acid-1H-benzimidazole-2-methyl ester to gambogic acid is 2.05: 1) diluted by DMF (dimethyl formamide) after 25-30 min, stirring at room temperature for reacting overnight, pouring the reaction liquid into water, and sequentially extracting with ethyl acetate for 3 times, wherein each time is 14 mL; the organic phases are combined and dried by anhydrous magnesium sulfate, the magnesium sulfate is filtered, and the ethyl acetate phase is concentrated and evaporated to dryness at the temperature of 43 ℃ under reduced pressure to obtain a yellow crude product; the product was separated by column chromatography (ethyl acetate: dichloromethane ═ 1: 11) to give a gambogic acid derivative. The structural formula is as follows:
compared with the gambogic acid nuclear magnetic hydrogen spectrum, the more peaks are as follows:1H NMR(400MHz，DMSO-d6)δ：11.01(s，1H，O＝C-NH)，7.89～7.79(d，4H，O＝C-NH-Ar-H)，5.51(s，2H，O＝C-O-CH 2)，5.09(s，1H，N＝C-NH)，7.63(d，2H，C＝N-Ar-H)，7.29(m，2H，Ar-H)。
compared with the gambogic acid nuclear magnetic carbon spectrum, the more peaks are as follows:13C NMR(300MHz，DMSO-d6)δ：161.4，141.7，141.0，137.7，130.0，124.2，122.8，120.6，114.1，64.3。
the above results indicate the successful preparation of gambogic acid derivatives.
Example 1:
a feed additive comprises the raw material components of, by weight, 3 parts of Iris pallida root extract, 2 parts of beta-L-aspartyl-L-lysine, 2 parts of gambogic acid derivative, 1.2 parts of oligosaccharide, 0.7 part of selenium methionine, 0.5 part of vitamin C phosphate and 0.02 part of digestive enzyme.
The preparation of the feed additive, the components are mixed and stirred evenly to obtain the feed additive.
Example 2:
a feed additive comprises the raw material components of 2 parts of Iris pallida root extract, 2 parts of beta-L-aspartyl-L-lysine, 1 part of gambogic acid derivative, 0.8 part of oligosaccharide, 0.6 part of selenium methionine, 0.4 part of vitamin C phosphate and 0.03 part of digestive enzyme by weight.
The preparation of the feed additive was the same as in example 1.
Example 3:
a feed additive comprises the raw material components of, by weight, 4 parts of Iris pallida root extract, 3 parts of beta-L-aspartyl-L-lysine, 3 parts of gambogic acid derivative, 1.8 parts of oligosaccharide, 0.75 part of selenium methionine, 0.6 part of vitamin C phosphate and 0.01 part of digestive enzyme.
The preparation of the feed additive was the same as in example 1.
Example 4:
a feed additive comprises the raw material components of 3 parts of a vetiver iris root extract, 2 parts of beta-L-aspartyl-L-lysine, 1.2 parts of oligosaccharide, 0.7 part of selenium methionine, 0.5 part of vitamin C phosphate and 0.02 part of digestive enzyme.
The preparation of the feed additive was the same as in example 1.
Example 5:
a feed additive differed from example 4 in that: also comprises 0.04 weight part of (5E) -5- (4-ethoxy benzylidene) -2-mercapto-1, 3-thiazole-4 (5H) -ketone.
The preparation of the feed additive was the same as in example 4.
Example 6:
a feed additive differed from example 1 in that: also comprises 0.04 weight part of (5E) -5- (4-ethoxy benzylidene) -2-mercapto-1, 3-thiazole-4 (5H) -ketone.
The preparation of the feed additive was the same as in example 1.
Comparative example 1:
a feed additive differed from example 1 in that: not containing the extract of Iris pallida root.
The preparation of the feed additive was the same as in example 1.
Test example 1:
1. characterization of nuclear magnetic resonance (1H and13C NMR)
weighing 3mg of sample, dissolving the sample in DMSO to prepare a sample solution, placing the sample solution in a nuclear magnetic resonance instrument, and measuring. The operating conditions of the instrument are as follows: AVANCE III 400 NMR spectrometer (Bruker) and AVANCE III 300 NMR spectrometer (Bruker). And analyzing the types and the amounts of hydrogen and carbon in the target product through the data of the hydrogen spectrum.
2. Test of bacteriostatic Effect
An oxford cup experiment is adopted to detect the bacteriostatic effect of the gambogic acid and the derivative on escherichia coli and staphylococcus aureus, and the gambogic acid is set as a positive control drug and the normal saline is set as a negative control drug. Respectively placing oxford cups on plates of escherichia coli and staphylococcus aureus which are fully coated with bacterial liquid, adding 80 mu L of 2mg/mL liquid medicine into the oxford cups, taking physiological saline as a negative control drug, culturing overnight in an incubator at 37 ℃, and observing the diameter of a bacteriostatic zone.
The test results are shown in table 1:
TABLE 1 inhibition zone diameter data
From the analysis in table 1, gambogic acid itself has no inhibitory effect on escherichia coli, but the prepared gambogic acid derivative has obvious inhibitory effect on escherichia coli; compared with the gambogic acid, the gambogic acid derivative has no obvious difference on the inhibition effect of the staphylococcus aureus.
Test example 2:
the coilia ectenes juvenile fish is fed with artificial feed through artificial domestication, after domestication is carried out for 25 days, the coilia ectenes juvenile fish is in a healthy and disease-free state, the body mass range is 2.11-3.64 g, the average body mass range is 2.6g, the body length range is 7.4-10.0 cm, the average body length range is 8.9cm, the coilia ectenes juvenile fish is randomly distributed to 9 cement ponds, 1000 tails of each cement pond, and then the coilia ectenes juvenile fish is randomly divided into 3 groups, and each group is 3 times. Feeding different types of artificial feed by 3 groups respectively, feeding by a satiation method for 2 times (7: 30-8: 30, 14: 30-15: 30) every day; during the test, the average water temperature is 22.3 +/-1 ℃, and the dissolved oxygen is more than or equal to 5.5 mg.L-1Ammonium nitrogen is less than or equal to 0.02 mg.L-1Nitrite less than or equal to 0.2 mg.L-1The pH value is 8.0-8.5, and the salinity is 0.40-0.60. After the breeding for 60 days, weighing the body mass and the body length, and collecting blood, liver, pancreas and muscle samples.
And (3) feed setting: the basic feed and the feed additive (the weight portion is 0.32 percent) comprise the following blank groups: a basal feed. Wherein, the feed additives are the additives prepared in comparative example 1 and examples 1-6 and the commercial feed additives (control group).
TABLE 2 basal diet nutrition level (based on air drying sample)
| Nutritional levels | Moisture content | Dried substance | Coarse ash content | Crude protein | Crude fat | Calcium carbonate | Phosphorus (P) |
| Content (mg. g) | 481.24 | 551.32 | 120.63 | 249.31 | 103.72 | 22.36 | 0.87 |
Index and method of measurement
1. Biological index feeding
After the test for 60 days, randomly extracting 10 fishes from each pond, and measuring the body mass, the body length, the body height and the body thickness for measuring the fullness. The number of deaths during the trial was counted against daily management records and the total mantissas for each pool were calculated to calculate survival.
The fullness is equal to the weight of the fish body at the end of the period/the length of the fish body at the end of the period multiplied by 100 percent
Survival rate final total mantissa/initial total mantissa × 100%
The test results are shown in table 3:
TABLE 3 biological indicators
As can be seen from the analysis in table 3, compared with the blank group, the control group and the comparative example 1, when the feed additive prepared in example 1 is added into the feed, the fullness of coilia ectenes after 60 days is obviously improved, the effect of example 1 is better than that of example 4, and the effect of example 4 is slightly better than that of comparative example 1, which indicates that the presence of the iris tectorum root extract and/or the gambogic acid derivative in the feed additive can effectively improve the fullness of coilia ectenes, and the two are added simultaneously, so that the improvement effect of the compound application is better. The survival rate of the coilia ectenes juvenile fish bred by adding the feed additive prepared in the example 1 into the feed is obviously higher than that of the coilia ectenes juvenile fish bred in the example 4, and the effect of the example 4 is equivalent to that of the comparative example 1, which shows that the survival rate of the coilia ectenes juvenile fish can be effectively improved by the presence of the vetiver root extract and/or the gambogic acid derivative in the feed additive; meanwhile, the effect of example 5 is better than that of example 4, and the effect of example 6 is better than that of example 1, which shows that the improvement effect on the survival rate of the coilia ectenes juvenile fish is obviously enhanced by adding (5E) -5- (4-ethoxy benzylidene) -2-mercapto-1, 3-thiazole-4 (5H) -ketone.
2. Biochemical index of serum
Randomly collecting 10 tails of coilia ectenes juvenile fish from each pool, immediately wiping the tails with gauze, collecting blood with tail vein of disposable syringe, standing in refrigerator at 4 deg.C for 3 hr, and keeping at 4300 r.min-1Centrifuging at 4 deg.C for 12min, extracting serum, and storing in refrigerator at-20 deg.C for measuring biochemical index of each serum.
Determining the content of total protein and blood sugar by a biuret colorimetric method; measuring the blood sugar content by using a glucose oxidase method; the kit is purchased from Shanghai famous classics bioengineering company.
The test results are shown in table 4:
TABLE 4 serum Biochemical index test results
From the analysis in table 4, compared with the blank group, the control group and the comparative example 1, the feed additive prepared in the example 1 is added into the feed to feed the coilia ectenes juvenile fish, and the total protein content in the serum of the coilia ectenes is not significantly different, and the total protein content of the coilia ectenes in the examples 1 to 6 is not significantly different, which indicates that the total protein content in the serum of the coilia ectenes is not negatively influenced by the addition of the feed additive. Compared with a blank group, a control group and a comparative example 1, the feed additive prepared in the example 1 is added into feed to feed the coilia ectenes juvenile fish, the blood sugar content of the coilia ectenes juvenile fish is obviously improved, the effect of the example 1 is better than that of the example 4, and the effect of the example 4 is slightly better than that of the comparative example 1; generally speaking, the normal blood sugar content of the fish is in the range of 2.8-12.8 mmol.L-1Blood sugar rises in this range, indicating enhanced digestion and absorption by the fish; the feed additive shows that the feed additive can effectively enhance the digestion and absorption effects of the coilia ectenes due to the existence of the Iris pallida root extract and/or the gambogic acid derivative, and the coilia ectenes are added simultaneously, so that the effect of improving the compound use is better. Meanwhile, the effect of example 5 is equivalent to that of example 4, and the effect of example 6 is equivalent to that of example 1, which shows that the addition of (5E) -5- (4-ethoxy benzylidene) -2-mercapto-1, 3-thiazole-4 (5H) -ketone has no negative influence on the digestion and absorption effects of coilia ectenes.
3. Related enzyme activity and content test
Thawing liver sample, adding 10 times of 4 deg.C buffer solution, homogenizing in ice bath to obtain 10% homogenate at 4 deg.C 2500 r.min-1Centrifuging for 12min, and storing the supernatant in a refrigerator at-20 deg.C. Determining the lipase content in the liver; the assay kit was purchased from Nanjing Biotechnology Ltd. TestingThe results are shown in Table 5:
TABLE 5 Lipase content test results
| Sample (I) | Lipase content (g/L) |
| Blank group | 18.64±3.17 |
| Control group | 21.43±2.84 |
| Comparative example 1 | 22.21±3.26 |
| Example 1 | 38.61±2.94 |
| Example 2 | 37.77±3.07 |
| Example 3 | 38.23±2.51 |
| Example 4 | 29.18±2.98 |
| Example 5 | 30.10±3.19 |
| Example 6 | 39.72±4.48 |
As can be seen from the analysis in table 5, compared with the blank group, the control group and the comparative example 1, when the feed additive prepared in example 1 is added into the feed, the coilia ectenes juvenile fish is fed, the lipase content of the coilia ectenes after 60 days is obviously improved, the effect of example 1 is better than that of example 4, and the effect of example 4 is slightly better than that of comparative example 1, which indicates that the coilia ectenes juvenile fish can effectively utilize the feed due to the presence of the iris fasciolor root extract and/or the gambogic acid derivative in the feed additive, the fat synthesis is higher than that of decomposition, fat deposition exists in vivo, and the fullness and the specific growth rate are obviously improved; and the two are added simultaneously, so that the effect is improved by compounding.
Test example 3:
transport stress test
Transport experiment
Before the experiment begins, 6 water tanks are arranged, and 30 fishes are fed by a satiation method; experimental setup: experiment 1 group: basal feed + feed additive prepared in comparative example 1; experiment 2 group, basal feed + feed additive prepared in example 1; experiment 3 groups: basal feed + feed additive prepared in example 4; experiment 4 groups: basal feed + feed additive prepared in example 5; experiment 5 group: basal feed + feed additive prepared in example 6; control group: a basal feed.
Materials (10 fish) are taken after transportation for 6h, and another 10 fish is taken as a stress 0 point. The fast deep anesthesia is carried out by MS-222 with the concentration of 100mg/L, the body length, the body weight and other conventional biological data of each fish are measured before sampling, then gene expression analysis is carried out, partial tissues of the brain, the liver and the head and kidney are respectively taken and put into 1.5mL centrifuge tubes (RNAase free), 1mL of RNAioso Plus reagent is added into each centrifuge tube, and the mixture is stored in a refrigerator at the temperature of 20 ℃ below zero.
Total RNA was extracted and purified according to the method described in the RNAisso plus Specification, the integrity of RNA was checked by agarose gel electrophoresis, and then cDNA was synthesized by reverse transcription using the total RNA as a template according to the instructions in the TaKaRa reverse transcription kit and stored in a freezer at-20 ℃ for use.
The qRT-PCR method is adopted, cDNA is used as a template, beta-actin is used as an internal reference, and the expression of the gene in serum is analyzed. The 20. mu.L amplification reaction was as follows:
mixing, centrifuging, and placing on a fluorescent quantitative PCR instrument, wherein the reaction procedure is as follows: pre-denaturation at 95 ℃ for 4 min; then circulating for 45 times n under the conditions of 95 ℃ for 10s, 60 ℃ for 20s and 72 ℃ for 10 s. After the reaction is completed, the data are collated, as per 2-ΔΔCtThe method of (3) processes the data.
TABLE 6 primer sequences used in RT-qPCR reactions
The test results are shown in fig. 1. The analysis in the figure shows that the expression level of each gene in the experiment 2 group is obviously lower than the zero point of emergency, and is equivalent to that of the experiment 1 group and the control group; compared with the experiment 1 group, after the treatment of the experiment 4 group, the expression level of Corticotropin Releasing Hormone (CRH) gene, Proopiomelanocortin (POMC) gene, Urotensin (UI) gene and cortisol receptor (GR) gene in the head and kidney is obviously increased, and is equivalent to the zero level of emergency, and the effect of the experiment 5 group is better than that of the experiment 2 group, which shows that the addition of (5E) -5- (4-ethoxy benzylidene) -2-mercapto-1, 3-thiazole-4 (5H) -ketone in the feed additive, can effectively influence the expression of stress related genes such as CRH, UI, POMC, GR and the like, reduce the occurrence probability of inducing oxidative stress reaction, thereby effectively relieving oxidative stress caused by transport stress of the coilia ectenes and avoiding irreversible physiological changes of organisms caused by certain damage to tissues such as liver and the like. In addition, the expression levels of the UI and GR genes were higher than those of experiment 3 after the treatment of experiment 2, indicating that the presence of the gambogic acid derivative has a promoting effect on the expression of the UI and GR genes.
Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (9)
1. The use of a gambogic acid derivative in the preparation of a feed additive, the gambogic acid derivative having the structural formula shown below:
2. use according to claim 1, characterized in that: the gambogic acid derivative is prepared by grafting 4-aminobenzoic acid-1H-benzimidazole-2-methyl ester onto gambogic acid through acylation reaction.
3. Use of a gambogic acid derivative according to claim 3 for enhancing the survival and fullness of coilia ectenes.
4. A feed additive comprising: a gambogic acid derivative as claimed in claim 1.
5. A feed additive according to claim 4 wherein: the feed additive comprises the following raw materials, by weight, 2-4 parts of an iris tonkinensis root extract, 1-4 parts of beta-L-aspartyl-L-lysine, 0-4 parts of a gambogic acid derivative, 0.5-2 parts of oligosaccharide, 0.5-0.9 part of selenium methionine, 0.3-0.7 part of vitamin C phosphate and 0.01-0.04 part of digestive enzyme.
6. A feed additive according to claim 5 wherein: the raw material components are all in powder form, and the feed additive is obtained after the powder components are mixed and stirred uniformly.
7. A feed additive according to claim 5 wherein: in the extract of the iris tectorum, the tectoridin A is more than or equal to 33.2 percent and the tectoridin A is more than or equal to 29.3 percent.
8. A feed additive according to claim 4 wherein: the feed additive is added into the feed according to the weight percentage of 0.2-0.5%.
9. Use of a feed additive according to any one of claims 4 to 8 in the manufacture of a feed for enhancing the survival rate, digestive absorption and fullness of coilia ectenes larvae.
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