CN101623315B - Antibacterial immune double-effect compound Chinese herb for skin ulcerative syndrome of apostichopus japonicus - Google Patents
Antibacterial immune double-effect compound Chinese herb for skin ulcerative syndrome of apostichopus japonicus Download PDFInfo
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Abstract
The invention discloses an antibacterial immune double-effect compound Chinese herb for skin ulcerative syndrome of apostichopus japonicus. The herb comprises the following components: common andrographis herb, dyer woad leaf, honeysuckle and szechuan lovage rhizome; the weight ratio of the components is 2:1:3:2; and the herb is powered, wherein granularity is 200 mesh. The herb is orally taken to prevent and cure the skin ulcerative syndrome of apostichopus japonicus, wherein each kilogram feed is added with 20g of herb powder for the mild sea cucumber and each kilogram feed is added with 30g of herb powder for the critical sea cucumber. When in use, the herb is weighed according to required dosage and is fed after being evenly stirred with stichopus japonicus feed one time per day, wherein continues 20 days are a period of treatment. The Chinese herb is special for the skin ulcerative syndrome of apostichopus japonicus, which can be used for the young sea cucumbers and the adult sea cucumbers, has good protection effect during the peak period of the skin ulcerative syndrome, and has cure rate high to 80-90% and survival rate high to more than 85% for the apostichopus japonicus having the skin ulcerative syndrome within 2-3 weeks. The compound herb has a double effect for immunity and disease resistance, has no poison and side effect, convenient use, can reduce or replace the use of antibiotics, and is suitable for breeding the young sea cucumbers and controlling diseases during breeding and producing.
Description
Technical field:
The invention belongs to control and culture the special-purpose medicaments of Radix Morinae Bulleyanae major disease, is a kind of antibacterial immunity double-effect compound Chinese herbal medicine to the Radix Morinae Bulleyanae skin ulcerative syndrome.
Background technology:
At the beginning of 21 century, apostichopus japonicus culture is coastal flourish in northern China, and annual value of production surpasses 20,000,000,000 yuan, becomes the main body of the 5th sea-farming tide of China.Because the apostichopus japonicus culture scale is expanded rapidly, supporting technologies such as disease control relatively lag behind, add the operation lack of standardization in the production process, cause the disease problem to be on the rise, annual due to illness evil break out cause year economic loss above 3,000,000,000 yuan, wherein the harm that causes with Radix Morinae Bulleyanae " skin ulcerative syndrome " is the most serious, is the most important diseases that causes China's Radix Morinae Bulleyanae large tracts of land death.
Be treatment and prevention aquatic animal disease, the tradition method of preventing and treating is to use chemosterilant, antibiotic, and the history in existing more than 30 year; Because of life-time service, not only the Drug resistance of cause of disease is more and more obvious, and its drug residue also is on the rise, and causes disease to be difficult to cure, and aquatic product quality safety is affected; Vaccine also is effective way of prevention animal infectious disease, but defective such as targetedly strong, that sphere of action is little, wait invertebrates (as prawn, Radix Morinae Bulleyanae, shellfish etc.) effect limited for the low of main dependence nonspecific immunity system, and use operation also very inconvenient, be difficult to apply.
Before the present invention forms, mainly concentrate in the research of aspects such as epidemiology, nosetiology and histopathology about the report of culturing Radix Morinae Bulleyanae " skin ulcerative syndrome ".The inventor has taken the lead in carrying out the research of China's Stichopus japonicus disease, and has named breed Stichopus japonicus major disease " skin ulcerative syndrome " in 2004.Skin ulcerative syndrome is commonly called as " change skin disease ", and its cardinal symptom comprises: anorexia, shake the head, swollen mouth, row are dirty, mouthful turgescence canker, health atrophy, body surface large tracts of land ulcer, mortality rate reaches 80% in 1-2 week, its disease transmission is strong, involves wide.The pathogenic former multiformity that has of this disease, that has reported has 4 kinds, be respectively Vibrio splindidus (Vibrio splendidus), Pseudoalteromonas (Pseudoalteromona nigrifaciens), Vibro harveyi (Vibrio harveyi), pseudomonas putida (Pseudomonas putida), its disease symptom and main pathogen see table 1 for details.
Table 1: the disease symptom and the main pathogen of culturing Radix Morinae Bulleyanae " skin ulcerative syndrome ":
Up to now, domestic research report at Radix Morinae Bulleyanae " skin ulcerative syndrome " control medicine mainly is an antibiotic.Carry out the relevant report of oral medication with Chinese herbal medicine, rarely seen have Li Hua etc. " Chinese herbal compound preparation of treatment causative pathogen skin ulcerative syndrome of Apostichopus japonicus " (number of patent application: 200810012641.3), do not see that this prescription has effect and the relevant report that improves Stichopus japonicus immunity and pre-chemical defence skin disease.
Summary of the invention:
The object of the invention provides a kind of compound Chinese herb, solve the technical problem of its compound Chinese herb, make the use of this compound recipe can effectively improve Radix Morinae Bulleyanae immunity, and possess the function of the multiple pathogen of inhibition Radix Morinae Bulleyanae " skin ulcerative syndrome " simultaneously, improve the apostichopus japonicus culture survival rate.
The technical solution adopted in the present invention is:
Ingredient and proportioning are: ingredient is: Herba Andrographis, Folium Isatidis, Flos Lonicerae and Rhizoma Chuanxiong, medicine are Powdered; Medicine is by weight ratio: 2: 1: 3: 2.Granularity 200 orders.
Take mode: oral.
Drug dose is: oral prevention or treat light disease consumption, and the per kilogram feedstuff adds 20g medicated powder, and oral medication serious symptom consumption is that the per kilogram feedstuff adds 30g medicated powder.
Using method is: when oral prevention and treatment use, all take by weighing medicine according to above-mentioned consumption, throw something and feed after stirring with holothurian feed, throw something and feed every day 1 time, continuous 20 days is a course of treatment.
Compound recipe of the present invention has following characteristics:
1, the present invention is developing on the multiple former basis of carrying out folk prescription, compound recipe pharmacodynamic analysis and Stichopus japonicus immunization of causing a disease skin ulcerative syndrome, is having the immunity of the Radix Morinae Bulleyanae of raising, high-efficiency antimicrobial economic benefits and social benefits characteristics.
2, the Radix Morinae Bulleyanae that the present invention prepared " skin ulcerative syndrome " special-purpose medicaments is joined the phase and is become the ginseng phase all can use the children, can reach more than 80% the cure rate of ill Radix Morinae Bulleyanae, and survival rate reaches more than 85%.
3, use operational approach easy, only need medicine to be added to throw something and feed in the feedstuff in proportion to get final product, be applicable to that children's ginseng is cultivated and the control of breeding production disease.
4, the compound recipe composition adopts Chinese herbal medicine green, that have no side effect, and is cheap.Use the present invention to reduce or alternative antibiotic use, the commercial quality and the food safety of culturing Radix Morinae Bulleyanae had guaranteeing role.
Description of drawings:
Fig. 1: the variation of coelomic fluid neutral and alkali phosphatase (AKP);
sky-be expressed as blank, the normal feedstuff of only throwing something and feeding;
Portugal-be expressed as 1% β-1, the positive contrast of 3-glucosan;
High-as to be expressed as 3% compound Chinese herb;
In-be expressed as 1% compound Chinese herb;
Low-as to be expressed as 0.5% compound Chinese herb.
Fig. 2: the variation of acid phosphatase in the coelomic fluid (ACP).
The specific embodiment:
The present invention is developing on the multiple former basis of carrying out folk prescription, compound recipe pharmacodynamic analysis and Stichopus japonicus immunization of causing a disease skin ulcerative syndrome, is having antibacterial immunity economic benefits and social benefits characteristics.
Be described in detail technology contents of the present invention in conjunction with the accompanying drawings with embodiment below:
One, determining of the screening of ingredient of the present invention and proportioning:
1, the screening of medicine and processing:
According to herbal nature, select Rhizoma Atractylodis, Radix Et Rhizoma Rhei, Herba Portulacae, Radix Glycyrrhizae, Herba Andrographis, Folium Isatidis, Herba Houttuyniae, Radix Angelicae Sinensis, Flos Lonicerae, Cortex Acanthopancis, the Cortex Eucommiae, Herba Epimedii, Cortex Fraxini, Rhizoma Chuanxiong, Radix Bupleuri, Poria, Cortex Phellodendri, Radix Sanguisorbae, Radix Codonopsis, Galla Chinensis, Radix Isatidis and 24 kinds of Chinese herbal medicine dry powder of Radix Scutellariae for use, number consecutively is H-1, H-2, H-3......H-24.
Chinese herbal medicine dry powder respectively takes by weighing 5g after sieving by 200 orders, adds about 50 ℃ of distilled water respectively, and promptly 350mL soaked after 2 hours, was heated to boiling with big fire, and littler fire boils 90min; When being concentrated into 50mL, leach medicinal liquid with 8 layers of antiseptic gauze, use double-deck filter paper pressure filtration at last, promptly make the Chinese herbal medicine stock solution of 0.1g/mL concentration, 121 ℃ of sterilization 20min are standby.
With the 96 aseptic hole ELISA Plate (transparent plastic sheets that 96 holes are arranged, the big swept volume of every Kongzui is about 250 μ L) wherein 7 wells add and measure Chinese herbal medicine stock solution (0.1g/mL), (TSB is the pancreas peptone soybean broth culture medium with liquid TSB, available from Beijing overpass technology company limited) carry out 2 times of dilutions of going forward one by one, add 5 μ L, 0.5% red tetrazolium indicator in every then hole, be used to whether indicate bacterial growth.Shaking table is cultivated 6-8h in 27 ℃ of wet boxes, and liquid presents redness in its corresponding ELISA Plate hole of person that has the bacteria growing, and no change color person's minimum inhibitory concentration is the MIC value.
For influencing judgement person because of Chinese herbal medicine band color itself, dip in the mixed liquor of getting in the ELISA Plate hole with aseptic cotton carrier and coat that (TSB is the pancreas peptone soybean broth culture medium on the TSB flat board, both can be configured to the solid plate culture medium, also can be configured to fluid medium), directly take culture method to observe drug effect and minimum inhibitory concentration that whether bacterial growth judges medicine, see Table 2.The result shows that folk prescription Chinese herbal medicine H-5 (Herba Andrographis), H-6 (Folium Isatidis), H-9 (Flos Lonicerae) and H-14 (Rhizoma Chuanxiong) have good fungistatic effect to 4 strain Stichopus japonicus skin ulcerative syndrome pathogen, and its MIC value is respectively 6.25,7.81,5.86 and 3.52mg/L.
Show the minimum inhibitory concentration (mg/mL) of 2:24 kind folk prescription Chinese herbal medicine water decoction to 4 kinds of antibacterials:
2, inhibition zone is measured:
Cut-off is the flat pair of dish of about 90mm directly, injection is through the TSB of heating and melting 7-9mL, be evenly distributed at the bottom of the dish, place superclean bench, treat that it solidifies after, insert internal diameter 6.0 ± 0.1mm at each flat pair of medium branch of dish, external diameter 7.8 ± 0.1mm Oxford cup, pour 15-17mL again into, solidify back reuse sterilization tweezers the Oxford cup is taken out, just form culture medium flat plate with holes through the TSB of heating and melting.After treating that flat board is cooled to room temperature, with bacteria suspension (1 * 10
7Cfu/mL) with the evenly coating on perforated flat plate of sterilization cotton swab, in the hole, add 100 μ L Chinese herbal medicine stock solutions then, every kind of Chinese herbal medicine is done 2 parallel group simultaneously, measure record inhibition zone size (the inhibition zone size reflects the size of medicine fungistatic effect), get its meansigma methods and (average and to eliminate experimental error.Be the experiment basis of selecting 4 kinds of folk prescription Chinese herbal medicine).By the test of vitro inhibition " skin ulcerative syndrome " pathogen, from 24 kinds of Chinese herbal medicine, filter out 4 kinds of folk prescription Chinese herbal medicine of best fungistatic effect, promptly Herba Andrographis, Folium Isatidis, Flos Lonicerae, Rhizoma Chuanxiong see Table 3.
The inhibition zone of table 3:24 kind folk prescription Chinese herbal medicine water decoction is measured:
Annotate: because the hole of the cup-shaped one-tenth in Oxford is approximately 10mm, so antibacterial circle diameter≤10mm duration is 0.
And then design orthogonal test, see Table 4, compound Chinese herb H-5 (Herba Andrographis), H-6 (Folium Isatidis), H-9 (Flos Lonicerae), when H-14 (Rhizoma Chuanxiong) ratio is respectively 20g, 10g, 30g and 20g, its average diameter of inhibition zone reaches maximum respectively, is 15.25mm, 12.67mm, 13.50mm and 13.25mm.The best proportioning of compound recipe is H-5: H-6: H-9: H-14=2: 1: 3: 2.
Table 4: compound compatibility and fungistatic effect analytical table:
Annotate:
Expression MIC meansigma methods,
Expression antibacterial circle diameter meansigma methods.
Carry out extracorporeal bacteria inhibitor test again, the result proves that (H-5: H-6: H-9: H-14=2: 1: 3: 2) is obvious to the fungistatic effect of 4 kinds of pathogenic bacterium for this compound recipe, sees Table 5.
Table 5: compound Chinese herb is to the extracorporeal bacteria inhibitor test of 4 kinds of antibacterials:
"+" antibacterial "-" of representing not grow under this concentration is represented the antibacterial that grown under this concentration.
Two, the enforcement experiment and the effect of medicine:
1, immunoenhancement result detects test group:
Select healthy Stichopus japonicus individuality in Tian Yuan Aquatic product company limited holothruian cultures field, Yantai, Shandong in October, 2007, and the 30-90g/ head is raised in the white plastic case of about 35L size, adds water 20L.Every rearing in box is supported 6, raises 3 casees for every group, and test is divided into 6 groups, and the test group normal feedstuff is joined precious board children ginseng special compound feed for the benefit that Dalian Feng Yuan reaches the production of bait company limited.Go into Herba Andrographis, Folium Isatidis, Flos Lonicerae in every kg feedstuff, (mass ratio is Rhizoma Chuanxiong: 2: 1: 3: 2) compound Chinese herb, set 0.5%, 1%, 3%3 drug level gradient, each experimental group is its positive controls with 0.5%, 1% and 3% β-1,3-glucosan respectively, what only throw something and feed normal feedstuff is the blank group, and the forage volume of throwing something and feeding is thrown something and fed by 1% of Radix Morinae Bulleyanae body weight.The water source is the deep-well sea water, and average water temperature is 16 ℃.Morning, siphonage was inhaled the end, changed water once, threw something and fed once at dusk.
Support 1 week, not feeding in preceding 2 days, the back 5 days normal feedstuff of throwing something and feeding before the test temporarily.After on-test, per 5 days once sampling, fish for 4 Radix Morinae Bulleyanaes, each extracts the 0.3mL coelomic fluid in centrifuge tube at every turn, measures these 2 indexs just relevant with Radix Morinae Bulleyanae immunity of centrifuge tube mesenteron liquid caustic soda acid phosphatase (AKP) and acid phosphatase (ACP) that finish in 3 hours.As shown in Figure 1, each is organized reinforcing agent there were significant differences to the Radix Morinae Bulleyanae immunoenhancement result.The high dose interpolation group (3%) of compound recipe was since the 5th day, and AKP raises by a small margin, the 10th day, the utmost point is significantly higher than blank group (P＜0.01), and the 20th day peaking is blank 5.5 times of organizing, be 4 times of initial value (the 0th day), begin after 25 days to descend, but still be higher than blank group.0.5% performance of middle dosage group 1% and low dose group steadily.As shown in Figure 2,3% pair of Radix Morinae Bulleyanae coelomic fluid of high dose interpolation group ACP influence is the most obvious, in the time of the 10th day, the high dose interpolation is organized just, and the utmost point is significantly higher than blank group (P＜0.01), be reduced to subsequently and be lower than blank group, but peaking the 25th day the time is significantly higher than blank group (P＜0.05), be significantly higher than other test group ACP same period value simultaneously, the utmost point is significantly higher than initial value (P＜0.01).In dosage group and low dose group to compare difference in duration of test ACP vigor and blank group not remarkable.Show that by immunization experiment compound Chinese herb is adding the immunity that can significantly improve Radix Morinae Bulleyanae under 3% dosage, promptly add the 30g compound Chinese herb in the per kilogram feedstuff and can obtain best immune effect.
2, safety of medicine property testing:
Divide 2 experimental grouies to carry out.Add 20% compound Chinese herb (20g Chinese herbal medicine/80g feedstuff) in experimental group 1 feedstuff, add 25% Chinese herbal medicine (25g Chinese herbal medicine/75g feedstuff) in experimental group 2 feedstuffs, the matched group basic feedstuff of only throwing something and feeding.Each experimental group is established 3 repeating groups, and each repeating groups has 6 Radix Morinae Bulleyanaes (30-90g/ head).Support temporarily before the experiment and do not throw feedstuff in 2 days.During experiment the Chinese herbal medicine of aforementioned proportion is added in the fluid feedstuff (the fluid feedstuff Stichopus japonicus of throwing something and feeding can directly ingest), stir and soak the Radix Morinae Bulleyanae of throwing something and feeding after 2 hours, apostichopus japonicus culture is in the white plastic case.Determine the feed situation of Radix Morinae Bulleyanae by the 2nd day Radix Morinae Bulleyanae bait surplus and feces situation.Duration of test runs 15 days.The result shows: each experimental group Radix Morinae Bulleyanae does not have any unusual condition, and each ratio is added medicine and all Radix Morinae Bulleyanae do not caused toxic effect, and Radix Morinae Bulleyanae ingests normally, well-grown.
Conclusion: when oral medication carries out diseases prevention and treatment, add compound Chinese herb 30g/kg feedstuff, can improve Stichopus japonicus immunity, and Stichopus japonicus is had no side effect.
Three, using this compound Chinese herb treats 6 different breeding fields, area, Shandong trouble " skin ulcerative syndrome " Radix Morinae Bulleyanae.Effect such as following table 6:
The clinical pilot-scale experiment of table 6 compound Chinese herb control skin ulcerative syndrome
By a large amount of clinical prevention evidences: in the skin ulcerative syndrome high-incidence season, raise the prevention of throwing something and feeding with 2% companion, do not see morbidity, its preventive effect is good; Patients whose symptoms were less severe raise and throw something and feed with 2% companion, and the heavier person of symptom raises with 3% companion and throws something and feeds, and its cure rate is more than the 80-90% in the treatment 20 days of throwing something and feeding.Carried out the treatment pilot plant test in many plants in the clinical practice process, prevention effect is remarkable, has retrieved a large amount of economic losses for culturing unit.The present invention has started the precedent of utilizing Chinese herbal medicine to improve Stichopus japonicus immunity and effectively suppressing the skin ulcerative syndrome pathogen simultaneously, have immunity, disease-resistant economic benefits and social benefits effect, can reduce and replace antibiotic use, to solving major disease " skin ulcerative syndrome ", improve and culture survival rate and product quality, ensure that the breeding production stable and high yields has crucial meaning.
Claims (1)
1. the antibacterial immunity double-effect compound Chinese herbal medicine of a Radix Morinae Bulleyanae skin ulcerative syndrome is characterized in that its ingredient is: Herba Andrographis, Folium Isatidis, Flos Lonicerae and Rhizoma Chuanxiong; Medicine is by weight ratio: 2: 1: 3: 2; It is Powdered that medicine is, granularity 200 orders.
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| CN101623315B true CN101623315B (en) | 2011-01-26 |
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Families Citing this family (4)
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| CN102206595B (en) * | 2011-03-31 | 2012-12-05 | 国家海洋局第一海洋研究所 | Apostichopus japonicus antagonistic bacterium and its application |
| CN103463389A (en) * | 2013-08-30 | 2013-12-25 | 兴化市恒威生物技术有限公司 | Medicinal composition for treating skin ulceration syndrome of sea cucumber and preparation method thereof |
| CN104815249B (en) * | 2015-05-20 | 2018-04-27 | 山东亚康药业股份有限公司 | A kind of Chinese medicine preparation and preparation method for treating stichopus japonicus skin ulcer syndrome |
| CN108949615B (en) * | 2018-06-25 | 2020-05-05 | 青岛农业大学 | Microecological preparation for fermenting stichopus japonicus feed |
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