CN104222527A - Application method of betulinic acid - Google Patents
Application method of betulinic acid Download PDFInfo
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- CN104222527A CN104222527A CN201410520978.0A CN201410520978A CN104222527A CN 104222527 A CN104222527 A CN 104222527A CN 201410520978 A CN201410520978 A CN 201410520978A CN 104222527 A CN104222527 A CN 104222527A
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Abstract
The invention belongs to the technical field of livestock breeding, and specifically discloses application of betulinic acid to the preparation of a preparation for preventing and curing the diarrhea of livestocks, particularly the weaned piglets. The betulinic acid is added into the weaned piglet feed in a dosage of 0.1-0.5%, and is capable of remarkably reducing the diarrhea rate of the weaned piglets, improving the intestinal mucosal form, protecting the intestinal mucosal barrier, effectively improving the integral disease prevention capability of the piglets and improving the daily gain.
Description
Technical field
The invention belongs to the technical field of cattle breeding, be specifically related to betulinic acid and preparing prevention and therapy domestic animal, the especially purposes of the preparation of diarrhea of weaned piglets.
Background technology
In present level intensive culture, grice diarrhoea is the disease that common harm is very large.This disease directly causes piglet survival ratio decline, poor growth, arrest of development and become cad pig, even dead, affects the speed of growth and the feed conversion rate of whole feeding period, and final cultivation industrial belt of giving carrys out heavy economic losses.According to statistics, the piglet death that China causes because suffering from diarrhoea every year accounts for 39.8% of the dead sum of piglet.The average attack rate of grice diarrhoea is 46.5%, and the death rate is 15%, and the direct economic loss caused is up to 1,000,000,000 yuan.The reason of post-weaning diarrhea in piglet is a lot, mainly contains indigestion, immunity degradation, EI, the infecting and wean nutrition and environmental stress etc. afterwards of enteropathogenic Escherichia coli and other pathogenic bacteria.For a long time, the control of this disease depends on antibiotic and chemicals solves, and antibiotic abuse causes the medicament residue etc. in drug resistance generation, endogenous infection and suprainfection, immunity of organisms decline and food to bring huge threat to human health.Therefore, finding effective Substitutes For Antibiotic is major issue urgently to be resolved hurrily.
Betulinic acid belongs to lupinane type pentacyclic triterpene material, is distributed widely in as in the various plants such as Rhamnaceae, Myrtaceae and Betulaceae, and its amount is general very low, higher with Japanese birch bark amount.The treatment that birch-bark can be used for the diseases such as yellow subcutaneous ulcer, acute mastitis, scabies is recorded in Compendium of Material Medica.As far back as 19th century, Japanese birch bark is just placed in tea and Other Drinks by American and drinks; White birch myron is called " natural beer " and " forest beverage " by European.Nineteen ninety-five finds that betulinic acid has extremely strong selecting cell toxicity to MC, and suppress copying of HIV in H9 lymphocyte, its sphere of action almost affects each link of viral lifecycle.Because betulinic acid has superior antitumor and HIV-resistant activity, cause people to the extensive research of betulinic acid and concern.Study discovery subsequently, betulinic acid has the multiple stronger biologically actives such as immunological regulation, anti-oxidation stress, anti-inflammatory, and the rarely seen report of the application of betulinic acid in grice diarrhoea.It is reported, the responsive intensity of betulinic acid to cancer cell is about Normocellular 10 times, when betulinic acid dosage is up to 500mg/kg, animal does not have obvious poisoning symptom, and betulinic acid extensively derives from natural various plants, illustrating that betulinic acid has safety, not easily produces drug resistance, there is not the advantages such as medicament residue, is one of desirable Substitutes For Antibiotic.
Summary of the invention
A kind of betulinic acid is the object of the present invention is to provide to prepare prevention and therapy domestic animal, especially the purposes of diarrhea of weaned piglets preparation, use betulinic acid safely, have no drug resistance, with strong points, effectively can control diarrhea of weaned piglets, have the immunity and production performance effect that improve body concurrently, alternative antibiotic carries out feeding and interpolation simultaneously.
The application process of betulinic acid, described betulinic acid is for the preparation of the antidiarrhoea formulation of domestic animal.Described antidiarrhoea formulation can also improve the daily gain of domestic animal, reduces feedstuff-meat ratio.
Described domestic animal is pig, especially weanling pig.
By direct for described betulinic acid feeding domestic animal or add domestic animal to basal diet in feeding.
Described domestic animal is pig, especially weanling pig.
The mass ratio added to by described betulinic acid in weanling pig basal diet is 0.1 ~ 0.5%.Preferred 0.1-0.2%.
The preparation process of described betulinic acid is: from Japanese birch bark, extract betulinol, after intermediate product betulonic acid is prepared in Jones reagent oxidation, synthesizes betulinic acid with sodium borohydride reduction.
The content adopting HPLC to measure betulinic acid is 96.53%.
The betulinic acid used in the present invention utilizes existing technology path to prepare, from Japanese birch bark, extract betulinol, after intermediate product betulonic acid is prepared in Jones reagent oxidation, synthesizes betulinic acid with sodium borohydride reduction.(bibliography: Yi Jine, Wen Lixin, Yuan Liyun, Obminska-MrukowiczBozena, Yuan Hui. the extraction of betulinol and betulinic acid study on the synthesis in silver birch. Agricultural University Of Hunan's journal, 2010,36 (5): 574-580.)
The advantage that the present invention has:
The betulinic acid that the present invention adopts, has various biological activity, and has disease-proof functions concurrently, not only preventable disease but also can improve breeding performonce fo animals, and betulinic acid not easily remains in animal body, not easily produces drug resistance, guarantees the quality of livestock products.
1. Growth Performance of Weaning Piglets can be improved: betulinic acid additive group, compared with control group (not having additive), improves daily gain, reduces feedstuff-meat ratio.
2. obviously suppress diarrhea of weaned piglets: betulinic acid additive group is compared with control group (not having additive), and diarrhea rate significantly reduces;
3. can promote intestinal regeneration: obviously improve weanling pig Mucosal Morphology in Small, alleviate intestinal villi atrophy, betulinic acid additive group significantly improves duodenal height of naps, significantly reduces Crypt depth, improves the ratio of height of naps and Crypt depth.
Accompanying drawing explanation
Fig. 1 is the impact of betulinic acid on Growth Performance of Weaning Piglets;
Compared with blank group, " * " significant difference (P<0.05);
Fig. 2 is the impact of betulinic acid on weanling pig feed-weight ratio;
Compared with blank group, " * " significant difference (P<0.05), " * * " difference is extremely significantly (P<0.01);
Fig. 3 is the impact of betulinic acid on grice diarrhoea rate;
Compared with blank group, " * * " difference extremely significantly (P<0.01);
Fig. 4 is betulinic acid on the impact of weanling pig small intestinal mucosa height of naps and Crypt depth;
Compared with blank group, " * " significant difference (P<0.05);
Fig. 5 is betulinic acid on the impact of weanling pig small intestinal mucosa height of naps and Crypt depth ratio.
Compared with blank group, " * * " difference extremely significantly (P<0.01).
Detailed description of the invention
The betulinic acid that the present invention uses belongs to pentacyclic triterpenoid, derives from Japanese birch bark, from Japanese birch bark, extracts betulinol, after intermediate product betulonic acid is prepared in Jones reagent oxidation, synthesizes betulinic acid with sodium borohydride reduction.Concrete preparation method is as follows: the bark collecting white birch wood, through 60 DEG C of oven dry, is housed in dark, dry place.Semi-synthesis method is adopted to prepare betulinic acid.1) from Japanese birch bark, betulinic acid is extracted: take Japanese birch bark powder, hot reflux 3h is added with methyl alcohol 70 DEG C, drying under reduced pressure, adds 2moL/L NaOH and stirs, and collects subnatant, evaporated under reduced pressure also uses ether dissolution, add appropriate water fully to shake up, collect upper liquid, drying under reduced pressure, silica gel column chromatography, drying under reduced pressure obtains betulinol sterling.2) betulonic acid is adopted to be the method that intermediate synthesizes betulinic acid: with acetone solution betulinol rapid stirring, after being cooled to 5-10 DEG C, dropwise add the Jones reagent (birch Chun ︰ Jones reagent=1 ︰ 6) of brand-new, 20 DEG C of reaction 3h, cross and filter blackish green precipitation, evaporated under reduced pressure acetone, raffinate is extracted with ethyl acetate, washing, 10%NaHCO
3washing, the anhydrous MgSO of organic phase
4drying, obtains faint yellow solid (betulonic acid crude product) ether dissolution, and flash column chromatography is separated, and drying under reduced pressure, obtains betulonic acid sterling.Use sodium borohydride/oxolane (NaBH again
4/ THF) reduction, gained solid be dissolved in methyl alcohol and boil 15-20min, cooling the betulinic acid sterling that is recrystallized to obtain.The content adopting HPLC to measure betulinic acid is 96.53%.
By following pharmacodynamic experiment, the present invention proves that betulinic acid may be used for the diarrhoea improving weanling pig, and improve the growth performance of weanling pig.
Example 1:
Healthy male KM mouse 30,4-5w, SPF level, body weight (20 ± 2) g, credit number: (Hunan) 2009-0004, feed is the common incubation material of M02 mouse, and mouse and feed are purchased from Hunan Si Laike Jing Da animal used as test company.Mouse is placed in (22-25) DEG C, relative humidity is in the feeding room of 50% ~ 70%, after raising 1w, be divided into 3 groups at random, i.e. blank group (A group), endoxan group (B group, intestinal mucosal injury model), betulinic acid and cyclophosphamide combined group (C group, betulinic acid press 0.5mg/kg b w to).Betulinic acid is suspended in the soluble starch of 1% by C group, and adopt the mode administration of gavage, A group and B group only gavage the soluble starch of 1%, all by 20ml/kg bw to, 1/d, continuous 14d, observation clinical manifestation and changes of weight.Last give with 1h after modeling, B group and C group mouse peritoneal inject endoxan (50mg/kgbw), and A group injects isopyknic sterile saline.Raise and terminate, water 12h is can't help in mouse fasting, and de-cervical vertebra is put to death, and draws materials and carries out indices detection.
1) betulinic acid is on the impact of Mouse Weight, thymus index and spleen index
Betulinic acid is on the impact of Mouse Weight, thymus index and spleen index in table 1, and compared with blank group, endoxan group body weight and spleen index decrease, but difference is not remarkable; But thymus index obviously reduces (P<0.05), prompting endoxan makes normal mouse immunologic function be suppressed, and causes body weight to decrease; Compared with endoxan group, the body weight of betulinic acid group mouse, thymus index and spleen index all obtain recovery to a certain extent, and thymus index significant difference (P<0.05), show that betulinic acid can improve the immunologic function of hypoimmunity mice.
Table 1 betulinic acid is on the impact of Mouse Weight, thymus index and spleen index
Compared with blank group, " * " significant difference (P<0.05); Compared with endoxan group, " # " significant difference (P<0.05).
2) betulinic acid is on the impact of IL-2 and IL-10 content in mouse intestinal
It is immune response that Thl/Th2 system keeps stable, and the ratio of conventional IL-2/IL-10 represents.Betulinic acid affects in table 2 enteron aisle Thl/Th2, and compared with blank group, endoxan group mouse intestinal mucosa IL-2/IL-10 ratio significantly reduces (P<0.05), and show that cellullar immunologic response is suppressed, immunologic balance is broken; Compared with model group, the IL-2/IL-10 ratio of betulinic acid group enteron aisle significantly raises, and prompting betulinic acid by raising Th1/Th2 level, can reverse its drifting state, thus recovers mucous membrane balance.
Table 2 betulinic acid is on the impact of IL-2 and IL-10 content in mouse intestinal
Compared with blank group, " * " significant difference (P<0.05), " * * " difference is extremely significantly (P<0.01); Compared with endoxan group, " # " significant difference (P<0.05), " ## " difference extremely significantly (P<0.01).
3) betulinic acid is on the impact of mouse intestinal mucosa morphology
Endoxan can cause small intestinal villous height to shorten, and height of naps/Crypt depth ratio declines, and in table 3, showing to digest and assimilate and resist foreign substance by small intestine, to invade function suppressed.Betulinic acid can improve the enteron aisle mechanical barrier damage that endoxan causes, and is conducive to maintaining the normal institutional framework of small intestine and physiological function.
Table 3 betulinic acid is on the impact of mouse intestinal mucosa morphology
Compared with blank group, " * " significant difference (P<0.05), " * * " difference is extremely significantly (P<0.01); Compared with endoxan group, " # " significant difference (P<0.05), " ## " difference extremely significantly (P<0.01).
4) betulinic acid is on the impact of mouse intestinal mucosa SlgA content
The change of the change display intestinal immunological barrier function that in small intestinal mucosa layer, secretory immunoglobulin A (SlgA) is measured.In table 4, compared with control group, the content of endoxan group mouse intestinal SIgA significantly reduces (P<0.01), and prompting endoxan causes mouse intestinal mucosal immune barrier injury; Compared with endoxan group, betulinic acid can significantly improve SIgA content (P<0.01), and the SIgA secretion of prompting betulinic acid to intestinal mucosal injury mouse has improvement result, plays certain protective role to intestinal immunological barrier.
Table 4 betulinic acid is on the impact of mouse intestinal mucosa SlgA content
Compared with blank group, " * * " difference extremely significantly (P<0.01); Compared with endoxan group, " ## " difference extremely significantly (P<0.01).5) betulinic acid is on the impact of mouse intestinal anti-oxidation function
As shown in Table 5, compared with blank group, endoxan group small intestine SOD is active significantly to decline, and MDA content significantly raises (P<0.01), and prompting endoxan reduces the oxidation resistance of mucous membrane of small intestine; Compared with endoxan group; betulinic acid group mucous membrane of small intestine SOD is active significantly to be strengthened, and MDA content significantly reduces (P<0.01), illustrates that betulinic acid has oxidation resistance; can obviously alleviate the oxidative stress caused by endoxan, enteron aisle is played a protective role.
Table 5 betulinic acid is on the impact of mouse intestinal SOD and MDA level
Compared with blank group, " * * " difference extremely significantly (P<0.01); Compared with endoxan group, " ## " difference extremely significantly (P<0.01).
Example 2:
Feeding experiment selects Du × large × long three way cross weanling pig of 90 of same nursery house 28 age in days wean, body weight (8.4 ± 0.72) kg, be divided into 3 experimental group at random, each experimental group 3 repetition, wherein A group is blank group (basal diet, do not add any medicine), B group is betulinic acid group (basal diet+0.1% betulinic acid); C group is antibiotic group (basal diet+50ppm terramycin+50ppm aureomycin), experimental period 21d.Test and within first 1 week, comprehensively clean pig house, sterilize, before test, the expelling parasite of each group once, adopts closed management model management colony house.Divide early, middle and late concentrating for three times to throw something and feed, have a small amount of feed remaining as suitable with hopper, every day progressively increases.Adopt automatic drinking bowl freely to drink water, clear up ight soil in time, keep colony house well-ventilated.Ground keeps dry, and every day checks ight soil.
1) betulinic acid is on the impact of piglet growth performance
As can be seen from Fig. 1 and Fig. 2, during off-test, compared with blank group, betulinic acid can significantly improve net gain and the Average weight increasing a day of 60 age in days piglets, improves significantly (P<0.05) to feed-weight ratio; Illustrate that betulinic acid can improve the production performance of piglet, improve food conversion ratio.
2) betulinic acid is on the impact of grice diarrhoea
As can be seen from Figure 3, compared with blank group, betulinic acid obviously can reduce the diarrhea rate (P<0.05) of 60 age in days piglets; Illustrate that in the present invention, betulinic acid has Antidiarrheal efficacy, and effect is better.
3) betulinic acid is on the impact of weanling pig Mucosal Morphology in Small
As can be seen from Figure 4 and Figure 5, compared with blank group, betulinic acid obviously can raise height of naps degree/Crypt depth ratio, illustrates that betulinic acid can improve digesting and assimilating of small intestine and resist foreign substance and invades function, be conducive to maintaining the normal institutional framework of small intestine and physiological function.
Example 3:
Feeding experiment selects Du × large × long three way cross weanling pig of 90 of same nursery house 21 age in days wean, body weight (5.73 ± 0.41) kg, be divided into 3 experimental group at random, each experimental group 3 repetition, wherein A group is blank group (basal diet, do not add any medicine), B group is betulinic acid group (basal diet+0.2% betulinic acid); C group is antibiotic group (basal diet+50ppm terramycin+50ppm aureomycin), experimental period 28d.Test and within first 1 week, comprehensively clean pig house, sterilize, before test, the expelling parasite of each group once, adopts closed management model management colony house.Divide early, middle and late concentrating for three times to throw something and feed, have a small amount of feed remaining as suitable with hopper, every day progressively increases.Adopt automatic drinking bowl freely to drink water, clear up ight soil in time, keep colony house well-ventilated.Ground keeps dry, and every day checks ight soil.
The efficacy assessments result of the test of result of the test and example 2 without significant difference, therefore does not do deposited stating at this.
Above embodiments of the invention have been described in detail, but described content being only preferred embodiment of the present invention, can not being considered to for limiting practical range of the present invention.All equalizations done according to the present patent application scope change and improve, and all should still belong within patent covering scope of the present invention.
Claims (10)
1. the application process of betulinic acid, is characterized in that, described betulinic acid is for the preparation of the antidiarrhoea formulation of domestic animal.
2. the application process of betulinic acid according to claim 1, is characterized in that, described antidiarrhoea formulation can also improve the daily gain of domestic animal, reduces feedstuff-meat ratio.
3. the application process of betulinic acid according to claim 1 and 2, is characterized in that, described domestic animal is pig.
4. the application process of betulinic acid according to claim 3, is characterized in that, described domestic animal is weanling pig.
5. the application process of betulinic acid according to claim 1 and 2, is characterized in that, by direct for described betulinic acid feeding domestic animal or add domestic animal to basal diet in feeding.
6. the application process of betulinic acid according to claim 5, is characterized in that, described domestic animal is pig.
7. the application process of betulinic acid according to claim 6, is characterized in that, described domestic animal is weanling pig.
8. the application process of betulinic acid according to claim 7, is characterized in that, the mass ratio added to by described betulinic acid in weanling pig basal diet is 0.1 ~ 0.5%.
9. the application process of betulinic acid according to claim 8, is characterized in that, the mass ratio added to by described betulinic acid in weanling pig basal diet is 0.1 ~ 0.2%.
10. the application process of betulinic acid according to claim 1 and 2, it is characterized in that, the preparation process of described betulinic acid is: from Japanese birch bark, extract betulinol, after intermediate product betulonic acid is prepared in Jones reagent oxidation, synthesizes betulinic acid with sodium borohydride reduction.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105076716B (en) * | 2015-07-14 | 2018-08-17 | 湖南农业大学 | Application process of the betulinic acid as livestock fodder additives |
| CN108850614A (en) * | 2018-08-06 | 2018-11-23 | 杭州光启医疗科技发展有限公司 | A kind of disease-resistant fattening pannage of health care |
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| CN102550831A (en) * | 2012-02-20 | 2012-07-11 | 中国农业大学 | Application of cinnamic acid to preparation of product for improving growth performance of livestock and poultry |
| CN102960560A (en) * | 2012-11-23 | 2013-03-13 | 重庆优宝生物技术有限公司 | Composite acidifier for reducing post-weaning diarrhea rate |
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2014
- 2014-09-30 CN CN201410520978.0A patent/CN104222527B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102550831A (en) * | 2012-02-20 | 2012-07-11 | 中国农业大学 | Application of cinnamic acid to preparation of product for improving growth performance of livestock and poultry |
| CN102960560A (en) * | 2012-11-23 | 2013-03-13 | 重庆优宝生物技术有限公司 | Composite acidifier for reducing post-weaning diarrhea rate |
Non-Patent Citations (2)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105076716B (en) * | 2015-07-14 | 2018-08-17 | 湖南农业大学 | Application process of the betulinic acid as livestock fodder additives |
| CN108850614A (en) * | 2018-08-06 | 2018-11-23 | 杭州光启医疗科技发展有限公司 | A kind of disease-resistant fattening pannage of health care |
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