CN1853496B - Composition and method - Google Patents
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- CN1853496B CN1853496B CN2006100847225A CN200610084722A CN1853496B CN 1853496 B CN1853496 B CN 1853496B CN 2006100847225 A CN2006100847225 A CN 2006100847225A CN 200610084722 A CN200610084722 A CN 200610084722A CN 1853496 B CN1853496 B CN 1853496B
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Abstract
A companion pet diet meeting ordinary nutritional requirements for an aged pet and further comprising a sufficient amount of antioxidant or mixture thereof, to inhibit the deterioration of the mental capacity of an aged companion pet.
Description
The application is for dividing an application, and female case is to be October 30 calendar year 2001, application number " 01820994.7 " applying date, and denomination of invention is the application of " composition and method ".
Background of invention
Pet such as dog and cat face old and feeble problem.This also has certain embodiment in common saying.One of them is " old dog be can not learn new variety ".This common saying derives from following observation: along with the aging of dog, their intelligence and muscle power are same to be reduced.The intellection relevant with learning and memory as if reduce (Cummings BJ, Head E, Ruehl W, Milgram NW, Cotman CW 1996: dog is as old and feeble and dull-witted animal model: Neurobiology of aging 17:259-268).In addition, can manifest the behavior relevant with the intelligence change in aged animal changes.The reason that causes this type of ability reduction is a lot.
Shown that now the existence of at least a antioxidant of the level of signifiance in the diet of old pet suppresses the decline of old pet intelligence.
Summary of the invention
According to the present invention, a kind of pet diet that meets the general nutritional requirement of old pet is provided, and has wherein further contained a kind of antioxidant or its mixture of the old pet intelelectual deterioration of inhibition of effective dose.
Another aspect of the present invention is the method that suppresses old pet intelelectual deterioration, and this method comprises to a kind of antioxidant or its mixture of described pet fed with water flatfoot to finish this inhibition.
The present invention also provides the old pet diet that meets the general nutritional requirement of old pet, wherein further contain and be selected from following antioxidant: vitamin E, vitamin C, alpha-lipoic acid, 1-carnitine and any mixture thereof, its content is enough to suppress the decline of described old pet intelligence.
Another aspect of the present invention is the method that improves old pet intelligence, increases antioxidant or its mixture that improves intelligence comprising being enough to for described old pet feeding consumption.
In all these methods, need in the diet of these animals, add antioxidant or its mixture.
Detailed Description Of The Invention
The diet of giving old pet such as dog and cat feeding is the standard normal diet of feeding to the animal at this age.Be the typical food of at least 7 years old dog below.
Table 1
Component | Content |
Protein (% dry weight) | 19.5 |
Lipoic acid (% dry weight) | 10 |
Phosphorus (% dry weight) | 0.5 |
Sodium salt (% dry weight) | 0.2 |
The antioxidant or its mixture that add effective dose in described pet food can bring significantly and obvious variation to behavior, particularly intelligence in the old pet, especially shown in problem-solving ability.Term is old to refer generally at least 7 years old dog and at least 7 years old cat.
Dog and cat loss of mental capacity have been observed the several years.The forfeiture of this kind intelligence shows with some approach.For example, for dog, can be shown as disorient, indoorly urinate and defecate, the sleep-wake mode changes, reduce or change with people and the contacts of other pet and learning ability forfeiture and can't focusing one's attention on.These situations also can show in the cat.In dog and cat, there is not to find alzheimer's disease as the people suffered from.
For this type of intelligence forfeiture, a lot of theories have appearred.So far, the inventor does not find to suppress this type of intelligence forfeiture or can bring any diet action pathway as the optimum variation of intelligence that detects by target component really.
The inventor has obtained success in this regard.By using the diet of its invention, can show that the decline of old pet intelligence is suppressed, and increase as this ability shown in the ability to solve problem test.This species impoverishment of intelligence can reverse basically.Need the intelligence of the old pet of this treatment to improve.Show as memory and learning ability, deal with problems and to improve.Whole intellectual level is improved.Descend and to slow down with old relevant cognitive ability.For cognitive disorder syndrome, its progress can be slowed down in old dog, and the clinical symptoms relevant with this syndrome can be controlled.Needing prevention and needing the pet of these components is purpose groups.
The component of finishing this effect in the diet is certain antioxidant or its mixture.Antioxidant is the material that can reduce free radical.The example of this type of material comprises food as ginkgo leaf, oranges and tangerines pulp, grape sauce, catsup, carrot and spinach (all these preferred dry), and other material such as beta carotene, selenium, Co-Q10 (ubiquinone), lutein, tocotrienols, isoflavones, S-adenosylmethionine, glutathione, taurine, N-acetyl cysteine, vitamin E, vitamin C, alpha-lipoic acid, 1-carnitine etc.Vitamin E can with tocopherol or tocopherol mixture with and the form of multiple derivative such as ester use, as Vitwas E, succinate, palmitate etc.Preferred alpha form, but can comprise β, γ, δ form.Preferred d type, but racemic mixture is an acceptable.After pet is taken in, these forms and derivative will be brought into play vitamin E sample activity.Can in this diet, the form with ascorbic acid and derivative thereof use vitamin C, derivative such as synthos, cholesteryl salt, 2-monosulfate etc., after pet is taken in, they will bring into play vitamin C sample activity.They can be any forms, as liquid, semisolid, solid and heat-stable form.Alpha-lipoic acid can be used in the food with the form of the lipoic acid derivatives in alpha lipoic acid or the United States Patent (USP) 5621117, its racemic mixture, salt, ester or acid amides.In diet, the L-carnitine can be used, multiple derivative example hydrochloric acid salt, fumarate and the succinate of carnitine can be used, and acetylizad carnitine etc.
Consumption in diet all with percetage by weight (based on the dry weight) expression of this diet, calculates with active material, particularly calculates with dissociant.Maximum consumption can not bring toxicity.Can use at least about 100ppm or at least about the vitamin E of 150ppm.Operable preferable range is about 500 to about 1000ppm.Though optional, maximum generally is no more than about 2000ppm or about 1500ppm.For vitamin C, use at least about 50ppm, preferably at least about 75ppm, and more preferably at least about 100ppm.Can use nontoxic maximum.The amount of alpha lipoic acid can be at least about 25ppm, preferably about 50ppm, more preferably from about 100ppm.Maximum can be about 100ppm to 600ppm or the amount nontoxic to pet.Preferred range is that about 100ppm is to about 200ppm.For L-carnitine about 50ppm concerning dog, preferably about 200ppm, more preferably from about 300ppm is a useful minimum.For cat, the operable minimum of a value of L-carnitine is high slightly, according to appointment 100ppm, 200ppm, and 500ppm.Can use nontoxic maximum, for example, less than about 5000ppm.For dog, can use low amount, for example, less than about 5000ppm.For the dog preferable range is about 200 to about 400ppm.Mapping cat preferable range is that about 400ppm is to about 600ppm.
Can use the beta carotene of about 1-15ppm.
Can use about selenium of 0.1 to about 5ppm.
Can use lutein at least about 5ppm.
Can use tocotrienols at least about 25ppm.
Can use Co-Q10 at least about 25ppm.
Can use S-adenosylmethionine at least about 50ppm.
Can use taurine at least about 1000ppm.
Can use isoflavones at least about 25ppm.
Can use N-acetyl cysteine at least about 50ppm.
Can use cysteine at least about 50ppm.
Can use ginkgo biloba p.e at least about 50ppm.
Following is the raw material with high ORAC (oxygen radical absorbability).Fashionable when adding with 1% content (low component such as the total resultant of corn replaces 5% for ORAC), they have increased the 0RAC of whole diet, and have increased the edible blood plasma ORAC value that contains the animal of these component foods.If the amount with 1% is also united with other four kind of 1% component and to be accounted for 5% altogether, join in the diet, preferably can use any component of ORAC value greater than every gram dry weight Trolox equivalent of 25umol.
Spinach sauce
Catsup
Oranges and tangerines pulp
Grape sauce
Daucus carrot particles
Broccoli
Green tea
Ginkgo leaf
Corn gluten meal
Embodiment 1
All dogs are Beagle and are 7 years old or bigger.The nutrition composition of control group is identical with the disclosed diet of table 1 basically with tested diet.But control group food contains the 59ppm vitamin E and less than the vitamin C of 32ppm.Tested food contains 900ppm vitamin E and 121ppm vitamin C, the L-carnitine of 260ppm and the alpha lipoic acid of 135ppm.
Be assigned to before control group or tested group be rich in antioxidant diet group one group of basis of Beagle of giving 12 years old task of dealing with problems.The older animals matching is identical aspect study (reversal of identification) or memory (the non-coupling [DNMP] and the non-coupling [DNMS] to article that postpones to the position of delay).Compare the basic learning of two groups of dogs with the T check to reversal of identification study, DNMP and DNMS task.The result does not have conspicuousness.Therefore, the identification basis of dog is identical before diet is got involved.Begin this kind diet after about 1 month, first task of dealing with problems of arranging to dog is a mark identification learning task, and this is spatial attention test (Milgram etc., 1999, Milgram, N.W., Adams, B., Callahan, H., Head, E., Mackay, B., Thirlwell, C. , ﹠amp; Cotman (1999), C.W., dog is to the identification learning of mark, Learning ﹠amp; Memory, 6:54-61).
The mark identification learning requires object according to the approximate specific target of target selection.But initial study is based on the ability of dog learning objective identification mission.We find that at first the age depends on the complexity of task to the effect of identification learning, and we find that the target identification learning significantly reduces in the ability dog.
When the mark identification learning task of more tested enriched diet or control diet group older animals, has very high significant difference (p<02) between them.The comparison of enriched diet treated animal is made mistakes few by finishing the work according to the diet treated animal.In 40 unit, 6 animals of all of enriched diet group can meet the study standard, and have only 3 to be merely able to meet the study standard in 6 animals of control diet group.The mistake of being made when in addition, these 3 dogs are dealt with problems is more than the dog of accepting enriched diet.
After finishing the mark identification learning, the dog of control group or the tested diet group of antioxidant enrichment is accepted the test of odd number task.This task is shown 3 portions of foods that covered by 3 objects to dog.Wherein two objects identical and one be different.In order to obtain food, dog must be selected the target of odd number.The mistake of making when the dog of enriched diet group is learnt this task lack obviously than the dog of edible control diet that (all 4 odd numbers tests are marked in conjunction with p<.003).
Embodiment 2
Beagle (n=28) is accepted the size identification task earlier and is sorted according to the deviation with standard in these learning tasks.Then, be divided into 3 groups according to level according to ordering, and based on one of previous 3 kinds of diet of identification scoring Random assignment.All dog years age of registration in this test is all greater than 7 years old.To one of 3 kinds of different dried foodstuffs of the content of dog arrangement vitamin E, and beginning target identifying schemes.Content of vitamin E and other component see the following form 2.
Table 2
The food numbering | Vitamin E | Vitamin C | The L-carnitine | Lipoic acid |
1 | 799ppm | 114ppm | 294ppm | 135ppm |
2 | 172ppm | <32ppm | 42ppm | Do not add |
3 | 57ppm | <32ppm | 13ppm | Do not add |
This target identifying schemes is formed (target 0,1,2) by 3 experimental stages and is formed, and wherein requires dog to reach by standard (row 8/10 was correct in two days one, next three days average 7/10) before beginning next stage test.Allow dog day-to-day test in 40 days learn each stage 10 times.Repeat MANOVA and shown that diet has produced significant mass action (P<0.05) to the mistake of levels of the standard.The wrong summation of target 1+2 has provided the significantly regression slope of (P<0.05) to the regression analysis of the content of vitamin E of this diet, wherein the dog of the highest E diet is made a mistake minimum (average=65 times), and the dog of the minimum diet of E content make a mistake at most (comment equal=170 times).
Embodiment 3
30 adult dog of originating at random are used for this research.These dogs at least 10 months are big, do not have pregnancy, do not have lactication and on-test precursor overlap reason.With animal at random powder be 5 groups and carry out diet test, 3 every group male and 3 female.
Before the nursing phase in 2 weeks, all dog feeding control Food (0ppm dl-alpha-lipoic acid), this foods met or exceed all trophic levels (table 1) of recommending by U.S. feed control office worker association (AAFCO 2000).This is divided into 5 test group at random with them after feeding early stage, and wherein dl-alpha-lipoic acid target content (based on dry weight) is as follows respectively: 0ppm, 150ppm, 1500ppm, 3000ppm, 4500ppm.In all diet, contrast and alpha lipoic acid group are all adding vitamin E, content 600-1000 international unit, and with the content of 100-200ppm adding vitamin C.
Except water, tested food is sole source of nutrients.Connection with waking.Selected dog and weighing after the initial body weight, the ME that estimates based on food is that every dog is calculated quantity of food.Initial quantity of food calculate based on dog keep energy requirement (MER), and with the factor correction of consideration normal activity, calculate by following formula:
MER (kcal/ days)=1.6 * RER (rest energy demand)
Wherein: RER (kcal/ days)=70 * body weight (kg) 0.75
Weigh to dog weekly and adjust quantity of food as required so that give food with the optimal body weight that is enough to keep them.Optimal body weight is determined with 3 to 5 ratio.If dog after adjusting quantity of food, can not maintain body weight be reduced in initial body weight about 10% in, then from this research with its discharge.All detected values of record body weight or food intake.
Sample ground and with 5.0ml phosphate buffer (10mM sodium hydrogen phosphate, 2mM ethylenediamine tetra-acetic acid (EDTA), 0.9% sodium chloride, pH7.4) 4 extracted twice, 0.100 ± 0.001g sample.The extract of 250 μ L is joined in the 5ml glass centrifuge tube that has the Teflon cap.Add 15 μ L EDTA solution (100mM EDTA, the about 1M NaOH of usefulness are regulated pH to 7.8) and the freshly prepd 5mM dithioerythritols of 50 μ L (DTE).Stirring solution also at room temperature hatched 5 minutes.Add the 1M phosphoric acid of 10 μ L and the ether of 2.0mL then.Cover test tube under the room temperature, shake, and with 1500xg centrifugal 3 minutes.The ether layer is transferred in another 5ml glass centrifuge tube, uses the 1.5mL ether simultaneously again with the water layer extracting twice.All extracts of this sample are merged.Dried extract in the vaporized nitrogen device in the water-bath at room temperature then.At this moment, sample is closed the lid and freezing.
Then, the extract of drying is thawed and dissolve again with 70 μ L SDS/EDTA solution (0.11% lauryl sodium sulfate (SDS), 15mM EDTA, 0.9% sodium chloride) and the freshly prepd 1mMDTE of 5 μ L.Then, in each test tube, add freshly prepd sodium borohydride 50 μ L.Shaking test tube also at room temperature hatched 10 minutes.After 10 minutes, that sample is freezing down at-70 ℃.Before this solution thaws, add 20 μ L 2M HCl.After solution thawed, add 800 μ L 100mM carbonic hydroammonium.Agitation of solutions also adds the acetonitrile solution (mBBr) of the 100mM Momobromobimane of 5 μ L.Then, solution was in the dark hatched 90 minutes under the room temperature.
Hatch the back and extract, from sample, remove excessive mBBr and DTE derivative with the 1.5ml carrene.Water layer is placed HPLC.With 30% acetonitrile, 1% acetate, be adjusted to the mobile phase of pH3.95 and each to go up sample sad with 15 minutes separate sulfur of isocratic elution liquid wash-out with flow velocity 1.0mL/min with about 2M ammonium hydroxide.The density that this preparation hypothesis is extruded food is 1g/ml.
The aseptic collection blood sample to be carrying out complete blood count, 2 weeks and carry out the blood biochemical analysis the 0th, 28,56,84,112,140 and 168 day of this research before beginning.In addition, gathered the 15mL whole blood with isolated lymphocytes in the 0th, 28 and 84 day that intervenes at food.
The whole blood of anticoagulant heparin joined layering in the conical centrifuge tube of 50ml Accuspin (SigmaChemical) and add the phosphate buffered saline (PBS) (PBS) of equivalent., do not brake centrifugal 30 minutes of sample with 700g.The collecting monocytic cell layer is transferred in the conical centrifuge tube of 15ml, is suspended in again among the PB of 1-3ml, and centrifugal as before (washing for the first time).As described in washing for the first time, carry out the washing second time.At last, collecting cell also was suspended in the chloric acid (10%w/v), and freezing up to analysis down at-70 ℃.
Sample is transferred in the dry ice refrigerating box from-70 ℃ of household freezers.In refrigerated centrifuge centrifugal 5 minutes with 12000rpm.The analysis of 1 equal portions glutathione (GSH) is transferred in the conical test tube with supernatant.
Use by the Reed of Jones (Jones etc.) improvement and colleague's (Fariss etc.) thereof method derivatization the solubility in acid extract.
In brief, 150 μ l extracts or internal standard compound are joined the 1.5ml microcentrifugal tube, then add γ-glu-glu internal standard compound and the 50 μ l IAA of 20 μ l, then mix.Transferring pH value of solution with potassium hydroxide-saleratus working solution is about 10 (purples).In the dark solution is hatched 1 hour under the room temperature.Add the SangerShi reagent identical with cumulative volume, and with solution overnight incubation (20 hours) in the dark.
After hatching, with solution centrifugal 5 minutes, supernatant is transferred in another 1.5ml microcentrifugal tube with 12000rpm.200 μ l supernatants are joined in the automatic bottle of amber (amberautovial), and it has one 300 μ l inlet, and the top is analyzed to carry out HPLC with crimper is fixing.
Solvent and separation condition as mentioned above (Fariss, Jones).With respect to safe criterion quantitative assay GSH and GSSG level.Estimate derivatization efficient with γ-glu-glu as internal standard compound.
By the paired t check analysis of Windows SAS software clinical chemistry, hematology and body weight, its significant difference P<0.05 to the numeric ratio of baseline value.The mean value of putting each detection time is analyzed by one-sided ANOVA, significant difference P<0.05.Between group between the 84th day and the baseline value difference of GSH: GSSG analyze conspicuousness P<0.05 with one-sided ANVOA with Windows SAS software.
The result
Lipoic acid (ppm) concentration in the food of 7 continuous detecting (0,28,56,84,112,140,168 day) gained is in the detection sensitivity of estimating, and general conform to our instrument (table 1) of generation parameter.
The food intake data merit no attention.On average, most of animals were taken in more food at 6th month than testing when beginning in all groups.Lose weight (but 6 months the time this variations seem reverse) except some that originally take place in 4500ppm concentration group, weight data is not up to attention.
Routine physical examination do not find relevant with nutrition unusually or any evidence of dl-alpha-lipoic acid toxicity.All animals keep normal in whole research process in this test colony.Observing some animals in this research process vomits once in a while; But, do not observe draw this kind vomiting may be owing to the tendency of the inference of lipoic acid.In the highest group of content, be excluded in the 21st day in test, reason is to lose weight and leukocytosis.Leukocytosis in this animal does not solve when off-test yet, and suspection is owing to some other diseases cause.
For same group of dog, when the 28th, 56,84,112,140 and 168 day serum biochemistry numerical value and initial numeric ratio than the time, some significant differences merit attention, but, these all can not be counted as having the biology conspicuousness, because these numerical value are all in laboratory's term of reference or very approaching, and notice uniformity tendency in the some months.Comparison in each time period between control group and other processed group also discloses some significant differences; But these all can not be counted as having the biology conspicuousness, because these numerical value are all in laboratory's term of reference or very approaching, and do not have any tendency.
For same group of dog, when the 28th, 56,84,112,140 and 168 day hematology numerical value and initial numeric ratio than the time, some significant differences merit attention, but, these all can not be counted as having the biology conspicuousness, because these numerical value are all in laboratory's term of reference or very approaching, and notice uniformity tendency in the some months.Comparison in each time period between control group and other processed group also discloses some significant differences; But these all can not be counted as having the biology conspicuousness, because these numerical value are all in laboratory's term of reference or very approaching, and do not have any tendency.
GSH: GSSG ratio
Raising GSH in 84 days: the change list of GSSG ratio reveals the remarkable mass action (P=.024) of diet, wherein this ratio rising (table 2) of all enriched food groups.ANOVA shows that content is minimum and compares with basic diet group and to show significant difference with the highest group, but quantity increases maximum in the minimum content level.In other words, GSH in high-load and the minimum content group: the variation of GSSG ratio and same time period in basic food observed be changed significantly different.Can't detect the ratio of 4 points (1 control group, 3 processed group) at the 84th day, not fall GSSG because in these samples, detect.Like this, the reinforcement group may demonstrate higher GSH: the GSSG ratio, if this detection sensitivity is enough to detect the low-level GSSG at the 84th day.
Table 1
Content (ppm) | On average | Standard deviation | The object percentage composition |
0 | 24 | 17 | NA |
150 | 151 | 13 | 101 |
1500 | 1471 | 113 | 98 |
3000 | 2869 | 250 | 96 |
4500 | 4176 | 642 | 93 |
Table 2
Edible the 0th to 84 day GSH of dog that extrudes dl-alpha-lipoic acid in the feed: the variation of GSSG average proportions
Content (ppm) | Compare the 0th to 84 day GSH with basic food: the difference of GSSG ratio | N | The P value |
0 | -9.2±26 | 5* | NA |
150 | 70±20 | 6 | .003 |
1500 | 24±7 | 6 | .16 |
3000 | 10±4 | 4* | .46 |
4500 | 50±36 | 4* | .03 |
* in the contrast and 1 dog in the 4500ppm group do not detect GSSG at the 84th day, and 2 dogs did not detect GSSG at the 84th day in the 3000ppm group.
Carried out further observation for alpha lipoic acid.At the medium-term and long-term feed of diet alpha lipoic acid is safely and effectively.It has improved the glutathione (GSH) and oxidized glutathione (GSSG) ratio that reduces.The time of the long-term feed of alpha lipoic acid can continue minimum 1,2,3,4,5 or 6 months to 1,2,3,4,5 year in diet, in addition animal throughout one's life.Alpha lipoic acid need not carry out any special protection and can play a role as just sealing in diet, and needn't be present in the unit dosage forms of using in the medicine in the diet, for example, and tablet, pill, capsule etc.Lipoic acid Cmin in diet is about 25,50,75 or 100ppm.As long as maximum magnitude is lower than its toxic level, be lower than about 400,300 or 200ppm from start to finish.In general, be no more than about 6 or the 7mg/kg the weight of animals every day, preferably be no more than about 5.Alpha lipoic acid has improved the antioxidant defense function, and has improved the ability of animal anti-oxidative damage.Other an amount of antioxidant such as vitamin E and ascorbic the existence simultaneously also can reach all these effects.This shows that the effect of alpha lipoic acid has exceeded vitamin C and/or vitamin E.
Claims (21)
1. antioxidant or its mixture are used for suppressing the purposes of the diet of old pet intelelectual deterioration in preparation, wherein contain the alpha-lipoic acid at least about 25ppm in the diet.
2. the described purposes of claim 1, wherein said pet is a dog.
3. the described purposes of claim 2, wherein said dog at least 7 years old.
4. the described purposes of claim 1, wherein said pet is a cat.
5. the described purposes of claim 4, wherein said cat at least 7 years old.
6. the described purposes of claim 1 wherein contains the vitamin E at least about 100ppm in the diet, and content of vitamin E is measured by detecting diet.
7. the described purposes of claim 6 wherein contains the antioxidant that is selected from vitamin C, 1-carnitine or its mixture in the diet.
8. the described purposes of claim 1 wherein contains the antioxidant that is selected from vitamin C, 1-carnitine or its mixture in the diet.
9. the described purposes of claim 8 wherein contains the vitamin C at least about 50ppm in the diet.
10. the described purposes of claim 8 wherein contains the 1-carnitine at least about 50ppm in the diet.
11. antioxidant or its mixture are used for improving the purposes of the diet of old pet intelligence in preparation, wherein contain the alpha-lipoic acid at least about 25ppm in the diet.
12. the described purposes of claim 11, wherein said pet is a dog.
13. the described purposes of claim 12, wherein said dog at least 7 years old.
14. the described purposes of claim 11, wherein said pet is a cat.
15. the described purposes of claim 14, wherein said cat at least 7 years old.
16. the described purposes of claim 11 wherein contains the vitamin E at least about 100ppm in the diet, content of vitamin E is measured by detecting diet.
17. the described purposes of claim 16 wherein contains the antioxidant that is selected from vitamin C, 1-carnitine or its mixture in the diet.
18. the described purposes of claim 11 wherein contains the antioxidant that is selected from vitamin C, 1-carnitine or its mixture in the diet.
19. the described purposes of claim 18 wherein contains the vitamin C at least about 50ppm in the diet.
20. the described purposes of claim 18 wherein contains the 1-carnitine at least about 50ppm in the diet.
21. lipoic acid is used for improving the purposes of the diet of pet senescence phase anti-oxidative damage ability in preparation, described diet contains the lipoic acid at least about 25ppm.
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US24451000P | 2000-10-31 | 2000-10-31 | |
US60/244510 | 2000-10-31 | ||
US25344700P | 2000-11-28 | 2000-11-28 | |
US60/253447 | 2000-11-28 | ||
US09/922632 | 2001-08-06 | ||
US09/922,632 US20020076469A1 (en) | 2000-10-31 | 2001-08-06 | Composition and method |
US09/978,127 US20020115710A1 (en) | 2000-10-31 | 2001-10-16 | Composition and method |
US09/978127 | 2001-10-16 |
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