CN106942521B - Chinese herbal medicine preparation with stable medicine property and used for enhancing immunity and sterilizing prawn and method - Google Patents
Chinese herbal medicine preparation with stable medicine property and used for enhancing immunity and sterilizing prawn and method Download PDFInfo
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Abstract
A Chinese herbal medicine preparation with stable medicine properties for enhancing the immunity and sterilizing prawns and a method thereof are disclosed, wherein the Chinese herbal medicine preparation comprises 20-50% of astragalus polysaccharide, 20-50% of chlorogenic acid, 20-50% of allicin, 5-10% of sodium bisulfite and 3-6% of dimethyl-beta-thiothidine propionate by mass fraction. The method comprises pulverizing radix astragali, sieving, mixing with distilled water, decocting, concentrating the filtrate, adding ethanol until the ethanol content is 80%, and filtering overnight to obtain radix astragali polysaccharide; pulverizing Eucommiae cortex, sieving, mixing with ethyl acetate, stirring and reflux extracting in 65 deg.C water bath, mixing filtrates, and separating ethyl acetate to obtain chlorogenic acid. The preparation method is simple in preparation process, low in cost and convenient to apply, and the preparation can effectively improve the immune defense capability and the bactericidal effect of the prawns after production and application, effectively prevent and treat the diseases of the prawns, reduce the use of antibiotics and chemical drugs, provide technical support for the healthy and sustainable development of prawn culture and provide technical support for the protection of the ecological environment of a water area.
Description
Technical Field
The invention relates to a Chinese herbal medicine preparation with stable drug properties for enhancing immunity and sterilizing prawns and a method thereof, belonging to the technical field of aquatic product compound feed additives.
Background
At present, prawn culture is a strong supporting industry in aquaculture industry. With the increase of the prawn breeding density and the intensification degree, diseases caused by viruses, bacteria and the like become more serious day by day, and huge economic losses are caused to the prawn breeding industry. For these diseases, drug control is the most direct and effective method, but antibiotics and other chemical drugs are used for controlling breeding diseases for a long time, which results in a series of negative effects such as drug residues, generation of drug-resistant bacteria and the like. With the increasing importance of ecological environment and human health, the management and control of antibiotics and chemical drugs are becoming strict, and people are beginning to search for efficient and safe ways to improve disease resistance of aquaculture animals. The Chinese herbal medicine has the advantages of nature, high efficiency, low residue, difficult drug resistance and the like, and becomes a research hotspot for preventing and treating diseases of aquaculture at present, wherein the compound Chinese herbal medicine contains a plurality of effective components, and the components can act synergistically, so that the drug effect is greatly enhanced, and the compound Chinese herbal medicine has wide application prospect in preventing and treating the diseases of prawns.
Disclosure of Invention
The invention aims to provide a Chinese herbal medicine preparation with stable medicine property for enhancing the immunity and sterilizing the prawns and a method thereof.
In order to achieve the purpose, the invention adopts the following specific technical scheme:
a Chinese herbal medicine preparation with stable medicine properties for enhancing the immunity and sterilizing prawns is characterized by comprising 20-50% of astragalus polysaccharide, 20-50% of chlorogenic acid, 20-50% of allicin, 5-10% of sodium bisulfite and 3-6% of dimethyl-beta-thiofide by mass fraction.
A Chinese herbal medicine preparation with stable medicine property for enhancing the immunity and sterilizing prawns is characterized by comprising the following components: chlorogenic acid: allicin: sodium bisulfite: the mass ratio of the dimethyl-beta-thiodine propionate is 14: 14: 7: 2: 1.
the formula is mainly used for daily feeding, and can obviously enhance the immunity of the prawns.
A Chinese herbal medicine preparation with stable medicine property for enhancing the immunity and sterilizing prawns is characterized by comprising the following components: chlorogenic acid: allicin: sodium bisulfite: the mass ratio of the dimethyl-beta-thiodine propionate is 14: 14: 14: 2: 1.
the formula is more suitable for feeding the prawns before the emergence season of the bacterial infectious diseases, such as the middle and later culture periods, the bait feeding amount is continuously increased in the period, the water quality is gradually aged, residual bait and excrement discharge are increased, the number of pathogenic bacteria is greatly increased, the bottom of a shrimp pond is deteriorated, the water quality is difficult to control, and the diseases are easy to outbreak at the moment. The formula has more remarkable bactericidal effect.
The astragalus polysaccharide is extracted from astragalus, the chlorogenic acid is extracted from eucommia, and the astragalus polysaccharide is extracted by a water purification and precipitation method; chlorogenic acid is extracted with ethyl acetate. The allicin is a chemical synthetic product and is purchased from high biological technology limited company of Jinnan De; the sodium bisulfite is a chemical synthetic product and is purchased from Kailong chemical science and technology development Co., Ltd: the dimethyl-beta-thiothidine propionate is a chemical synthetic product and is purchased from Wuhanfeng bamboo forest chemical technology Co.
The invention has the advantages and beneficial effects that:
(1) the number of the blood cells of the litopenaeus vannamei is increased. In the non-specific immune system of prawn, blood cells play an important role, and are both the performer of cellular immunity and the provider of humoral immunity. Within 21 days of feeding the compound Chinese herbal medicine immunopotentiator, the number of the shrimp blood cells of the test group is obviously higher than that of the control group (P is less than 0.05), and the control group has no obvious difference.
(2) Improving the phagocytic activity of blood cells of the litopenaeus vannamei. The blood cells can wrap, destroy and eliminate foreign substances through phagocytosis, so that the phagocytic activity of the blood cells can be used for measuring the capability of the litopenaeus vannamei to resist pathogenic bacteria invaded from the outside. The phagocytic activity of the prawn blood cells in the test group is obviously higher than that in the control group (P is less than 0.05) within 21 days after the compound Chinese herbal medicine immunopotentiator is fed, and the control group has no obvious difference.
(3) Improving the activity of the phenol oxidase of the litopenaeus vannamei. The activity of the Litopenaeus vannamei serum phenol oxidase is obviously improved within 21 days after the compound Chinese herbal medicine immunopotentiator is fed, and the difference between each test group and a control group is obvious (P is less than 0.05).
(4) The activity of the Litopenaeus vannamei serum lysozyme is improved. Lysozyme is capable of destroying bacterial cell walls and is therefore considered an important humoral immune factor. The activity of the lysozyme of the Litopenaeus vannamei serum is obviously improved within 21 days after the compound Chinese herbal medicine immunopotentiator is fed, and the difference between each test group and a control group is obvious (P is less than 0.05).
(5) Improving the resistance of the litopenaeus vannamei to pathogenic vibrios. 7 days after the injection of the vibrio harveyi, the cumulative mortality rate of the prawns in the control group is 68.35%, the cumulative mortality rate of the prawns in the test group 1 is 33.35%, the cumulative mortality rate of the prawns in the test group 2 is 30%, and the difference between the test group and the control group is obvious (P is less than 0.05).
(6) The stability of the drug effect of the Chinese herbal medicine compound is improved. Chlorogenic acid is ester formed by caffeic acid and quinic acid, and the molecular structure of chlorogenic acid has ester bond, unsaturated double bond and three unstable parts of polyphenol, which can cause the content of effective components to be reduced due to oxidation. Meanwhile, the stability of the allicin is poor, the color is easy to deepen and even becomes sticky under the influence of external conditions, and the allicin is quickly decomposed to generate a plurality of allyl sulfides, so that the garlic processed products generate obvious garlic odor and green brown, and the biological function and the feeding efficacy are reduced. How to prevent oxidation and improve the stability of effective components is a key application factor influencing chlorogenic acid and allicin. At present, most of Chinese herbal medicine compounds related to chlorogenic acid or allicin only relate to evaluation of efficacy, and the stability of the efficacy is not mentioned. The sodium bisulfite has strong reducibility and can be used as an antioxidant, and the sodium bisulfite is added into the formula of the Chinese herbal medicine compound preparation, so that the stability of the Chinese herbal medicine compound preparation is improved, and the storage of the Chinese herbal medicine compound preparation is facilitated.
(7) The utilization rate of the compound is improved. Chlorogenic acid has sour and bitter taste, allicin has pungent taste, and the addition of these components will affect the palatability of the feed. The dimethyl-beta-thiothidine propionate (DMPT) has good feeding attraction effect on crustaceans, and the addition of low-concentration DMPT in the Chinese herbal medicine compound can greatly promote the ingestion of prawns and improve the utilization rate of the compound.
Experiments prove that the immunopotentiator provided by the invention can obviously enhance the immunity of prawns and improve the disease resistance of prawns; the enhancer selects three Chinese herbal medicines of astragalus polysaccharide, chlorogenic acid and garlicin to be scientifically matched, and is matched with two substances capable of improving the stability of the drug effect and promoting the ingestion, and the enhancer is added into the feed qualitatively and quantitatively, so that the stability of the drug effect and the standardized production are maintained; the use of antibiotics and chemical drugs can be replaced and reduced, the adverse effects of antibiotic abuse on the environment and the like are avoided, and the formula has higher ingestion promotion performance; the invention has simple preparation and lower cost, and can improve the economic benefit of the prawn breeding industry.
Drawings
FIG. 1 is a graph showing the results of the blood cell count of the prawn in example 2.
FIG. 2 is a graph showing the phagocytic activity of the hemocytes of the prawns in example 2.
FIG. 3 is a graph showing the results of the plasma dioxygenase activity of the prawns in example 2.
FIG. 4 is a graph showing the results of the plasma lysozyme activity of the prawns in example 2.
FIG. 5 is a graph showing the cumulative mortality of the prawns from the challenge test in example 2.
Detailed Description
Example 1:
the preparation method of the compound Chinese herbal medicine immunopotentiator and the basic feed comprises the following steps:
firstly, preparing active ingredients of Chinese herbal medicines, uniformly crushing flaky astragalus roots, sieving the crushed astragalus roots with a 80-mesh sieve, uniformly mixing the crushed astragalus roots with distilled water according to a mass-volume ratio of 1:10, decocting for 2 times and 2 hours each time, combining filtrates for 2 times, concentrating under reduced pressure, adding a proper amount of 95% ethanol to ensure that the final alcohol content is 80%, standing overnight, and performing suction filtration to obtain astragalus polysaccharides;
uniformly crushing sheet eucommia ulmoides, sieving with a 80-mesh sieve, uniformly mixing with ethyl acetate (containing 0.05mol/L hydrochloric acid) according to a mass-volume ratio of 1:8, stirring and refluxing for leaching for 3 hours on a 65 ℃ water bath, repeatedly extracting for 2 times, combining filtrates, and separating out ethyl acetate to obtain chlorogenic acid;
allicin, sodium bisulfite and dimethyl-beta-thiodine propionate are chemical synthetic products, which are purchased from Degao, Kailong chemical science and technology development Co., Ltd and Wuhanfeng bamboo forest chemical science and technology Co., Ltd respectively;
dissolving the Chinese herbal medicine extract, the allicin, the sodium bisulfite and the dimethyl-beta-thiothidine propionate in the proportion into water, uniformly spraying the mixture on the surface of a basic feed, wrapping the feed with fish oil, and airing to obtain the basic feed containing the compound Chinese herbal medicine immunopotentiator.
The reinforcing agent and the corresponding breeding feed are respectively prepared according to the preparation method, and two are obtained according to the two different specific proportions: an immunopotentiator comprises, by mass, 1g of astragalus polysaccharide, 1g of chlorogenic acid, 0.5g of allicin, 0.15g of sodium bisulfite and 0.07g of dimethyl-beta-thioridine added to each kilogram of feed; another immunopotentiator comprises astragalus polysaccharide 1g, chlorogenic acid 1g, garlicin 1g, sodium bisulfite 0.15g and dimethyl-beta-thiothidine propionate 0.07g added into each kilogram of feed.
The content of the effective components of the Chinese herbal medicines can be determined according to the method provided by the prior literature. The method for measuring the content of astragalus polysaccharide refers to: the determination of the content of astragalus polysaccharide in astragalus, which is a modern Chinese traditional medicine, No. 13 volume No. 07 in 2011; the determination method of the content of chlorogenic acid refers to: journal of Pharmaceutical Analysis, vol 1, 04, 2011; the allicin content determination method refers to: measuring the content of alliin and allicin in garlic by an HPLC method, wherein the content is measured in 2009, vol 35, No. 02; the content of the sodium bisulfite is measured by an oxidation-reduction titration method; the content of the dimethyl-beta-thiodine propionate is detected by adopting a sulfur gravimetric method.
Example 2:
influence of compound Chinese herbal medicine immunopotentiator on nonspecific immunity of Litopenaeus vannamei
In the experiment, the inventor feeds the two Chinese herbal medicine immunopotentiators prepared in the example 1 to the litopenaeus vannamei, and determines the nonspecific immunity index of the litopenaeus vannamei, so as to screen out the compound Chinese herbal medicine immunopotentiator for improving the nonspecific immunity function and the germ resistance of the litopenaeus vannamei.
1 materials and methods
1.1 materials
The Litopenaeus vannamei (Litopenaeus vannamei) for the test is purchased from a Litopenaeus vannamei breeding plant in Laoshan mountain area of Qingdao city, the average weight is 7.45 +/-0.41 g, and the components of the basic feed for the test are as follows: by mass fraction, 34% of fish meal, 20% of soybean meal, 16.4% of peanut meal, 22% of wheat flour, 5% of soybean lecithin, 0.6% of multivitamins and 2% of compound minerals. The experimental calculation shows that the nutrient components in the feed are as follows: 42.4% of crude protein, 7.2% of crude fat and 8.4% of ash in mass fraction.
1.2 methods
1.2.1 test design
After temporarily culturing litopenaeus vannamei for a test for 7 days, randomly dividing the litopenaeus vannamei into 3 groups, namely a control group, a test group 1 and a test group 2, wherein each group comprises 70 tails and each group comprises 3 parallel groups, feeding the litopenaeus vannamei in a 100L PVC bucket, and ventilating and feeding the litopenaeus vannamei for 24 hours. The control group was fed with basal feed, and the test 1 and 2 groups were fed with two immunopotentiator basal feeds prepared in example 1, respectively, 2 times per day, which were both full feeding amounts. The culture experiment lasts 21 days, and during the experiment, the water temperature, the salinity and the PH are respectively 25-27 ℃,34 per mill and 7.8-8.0.
1.2.2 measurement indices and methods
Blood was taken on days 0, 1, 3, 6, 10, 15, and 21 from the start of the test. Directly inserting sterilized No. 5 needle and 1mL syringe into the peripheral heart chamber of the posterior margin of the cephalic and thoracis of prawn for about 3mm for blood collection, and sucking 0.3mL improved precooled anticoagulant of Litopenaeus vannamei (0.34M NaCl,0.01M KCl,0.01M EDTA-Na2,0.01M HEPES, pH 7.45, osmotic pressure 780mOsm.kg in the syringe before blood drawing-1) And finally, the ratio of the anticoagulant to the hemolymph is 1:1, a part of hemolymph samples are subjected to the measurement of the number of blood cells and the phagocytosis rate, the rest hemolymph samples are centrifuged at 800 × g for 10min at 4 ℃, blue supernatant, namely a plasma sample, is taken, and the plasma sample is stored at-80 ℃.
The measurement of the number of blood cells was performed by counting under an optical microscope using a hemocytometer; the method for measuring phagocytic activity was carried out according to the methods of the prior art references: oxidative stress, DNA damage and antioxidant gene expression in the Pacific white shrimp, Lipopenaeus vannamei Whenexposition to access pH stress. The phenol oxidizing enzyme activity assay method can be referred to as: white shrim Lipopenaeus vannamei which received hot-water extract of Gracilaria tenuistica spent early recovery immunmunity after a Vibrio alisticity injection, Fish & Shellfish Immunology; the lysozyme activity was measured using a kit produced by Nanjing Biotechnology Ltd.
2 results
2.1 blood cell count
As can be seen from fig. 1: after the feed containing the compound Chinese herbal medicine immunopotentiator is fed for 21 days, the number of the blood cells of the litopenaeus vannamei is obviously increased, after the test is finished, compared with a control group, the number of the blood cells of the test group 1 and the number of the blood cells of the test group 2 are respectively increased by 14.2 percent and 16.5 percent, and the difference between the test group and the control group is obvious (p is less than 0.05). However, the increase in the allicin content in test group 2 did not further increase the number of blood cells compared to test group 1.
2.2 phagocytic Activity of blood cells
As can be seen from fig. 2: after the feed containing the compound Chinese herbal medicine immunopotentiator is fed for 6 days, the phagocytosis rate of the blood cells of the litopenaeus vannamei is obviously higher than that of a control group, but the difference between the test group 1 and the test group 2 is not obvious (p is more than 0.05). In the middle and later culture periods, the phagocytic capacity of the blood cells of the prawns in the test group 2 is increased more obviously and is obviously higher than that of the control group and the test group 1, and the fact that the high-concentration allicin is favorable for further improving the phagocytic capacity of the blood cells of the litopenaeus vannamei along with the extension of the culture time is shown.
2.3 phenol oxidizing enzyme Activity
As can be seen from fig. 3: after the feed containing the compound Chinese herbal medicine immunopotentiator is fed for 3 days, the activity of the serum phenol oxidase of the litopenaeus vannamei is obviously increased, and the activity of the serum phenol oxidase of the test group 1 and the test group 2 reaches the highest value on the 6 th day, and is obviously different from that of the control group (p is less than 0.05). From day 6 to the end of the experiment, the serum phenoloxidase activity of both test groups was slightly reduced, but still significantly higher than that of the control group. Throughout the test period, the difference between the activity of the serum phenol oxidizing enzyme in test group 1 and test group 2 was not significant. The results are combined, the astragalus polysaccharide, the chlorogenic acid and the garlicin added into the feed can improve the activity of the serum phenol oxidase of the litopenaeus vannamei, but the further improvement of the garlicin content can not continuously enhance the activity of the serum phenol oxidase of the litopenaeus vannamei.
2.4 lytic enzyme Activity
As can be seen from fig. 4: after the feed containing the compound Chinese herbal medicine immunopotentiator is fed for 3 days, the activity of the serum lysozyme of the litopenaeus vannamei is obviously increased and reaches the maximum value at the 10 th day, compared with a control group, the activity of the serum lysozyme of the test group 1 is increased by 2.05 times, the activity of the serum lysozyme of the test group 12 is increased by 2.23 times, but the lysozyme activity of each test group is slightly reduced at the later stage of the test and has obvious difference with the control group (p is less than 0.05).
2.5 Vibrio resistance
As can be seen from fig. 5: after 0.5 day of toxin attack, the shrimps in each group begin to die, each group dies 1-3 tails, the cumulative death number of the prawns in the control group is obviously higher than that of each experimental group by 1 day, the death number of each group is reduced by 3 days, and the death number of the prawns in each group is reduced and tends to be stable after 5 days. When the challenge test is finished, the cumulative mortality of the control group can reach 68.35 percent, the cumulative mortality of the test group 1 is 33.35 percent, and the cumulative mortality of the test group 2 is 30 percent, which shows that the vibrio resistance of the litopenaeus vannamei can be improved by feeding the feed added with the astragalus polysaccharide, the chlorogenic acid and the garlicin, and the vibrio resistance is improved along with the increase of the garlicin content.
Claims (2)
1. A prawn immunopotentiator is characterized by comprising the following raw materials in proportion: adding 1g of astragalus polysaccharide, 1g of chlorogenic acid, 0.5g or 1g of allicin, 0.15g of sodium bisulfite and 0.07g of dimethyl-beta-thiodine propionate into each kilogram of feed;
the preparation method of the astragalus polysaccharide comprises the following steps: uniformly crushing the flaky astragalus, sieving with an 80-mesh sieve, uniformly mixing with distilled water according to the mass volume ratio of 1:10, decocting for 2 times, each time for 2 hours, combining the filtrates for 2 times, concentrating under reduced pressure, adding a proper amount of 95% ethanol to ensure that the final alcohol content is 80%, standing overnight, and performing suction filtration to obtain astragalus polysaccharides;
the preparation method of chlorogenic acid comprises the following steps: uniformly crushing sheet eucommia ulmoides, sieving with a 80-mesh sieve, uniformly mixing with ethyl acetate containing 0.05mol/L hydrochloric acid according to the mass-volume ratio of 1:8, stirring and refluxing for leaching for 3 hours on a 65 ℃ water bath, repeatedly extracting for 2 times, combining filtrates, and separating out ethyl acetate to obtain chlorogenic acid.
2. A method for preparing a basal feed containing the prawn immunopotentiator according to claim 1, which is characterized by comprising the following steps: mixing astragalus polysaccharide, chlorogenic acid, allicin, sodium bisulfite and dimethyl-beta-thiothidine propionate according to the proportion of claim 1, dissolving in water, uniformly spraying on the surface of basic feed, wrapping with fish oil, and air drying to obtain the basic feed containing prawn immunopotentiator.
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| CN102274383A (en) * | 2011-07-19 | 2011-12-14 | 暨南大学 | Compound Chinese herbal medicine immunopotentiator for fish, and preparation method and application thereof |
| CN105124323A (en) * | 2015-10-13 | 2015-12-09 | 威海光洋生物科技有限公司 | Complex immunoenhancer for prawns |
| CN105648024A (en) * | 2016-01-29 | 2016-06-08 | 中国海洋大学 | Rapid screening method of compound Chinese-herbal-medicine immunopotentiator for prawns |
| CN105661105A (en) * | 2016-01-29 | 2016-06-15 | 中国海洋大学 | Compound Chinese herbal medicine immunopotentiator for prawns |
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| CN102274383A (en) * | 2011-07-19 | 2011-12-14 | 暨南大学 | Compound Chinese herbal medicine immunopotentiator for fish, and preparation method and application thereof |
| CN105124323A (en) * | 2015-10-13 | 2015-12-09 | 威海光洋生物科技有限公司 | Complex immunoenhancer for prawns |
| CN105648024A (en) * | 2016-01-29 | 2016-06-08 | 中国海洋大学 | Rapid screening method of compound Chinese-herbal-medicine immunopotentiator for prawns |
| CN105661105A (en) * | 2016-01-29 | 2016-06-15 | 中国海洋大学 | Compound Chinese herbal medicine immunopotentiator for prawns |
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