Embodiment:
(1) the clinical and experimentation (mental retardation abbreviates MR as) of treatment MR
1. clinical data MR70 name all is that intelligence, training school's case are educated in Hospital Attached to Liaoning Inst. of Traditional Chinese Medicine Pediatric Clinic and urban district, Shenyang in the period of the 1988-1990, male 40 examples, and women 30 examples divide observation group 50 example and matched group 20 examples at random.10 years old observation group's mean age, 11 years old matched group mean age.
2. diagnostic criteria:
1) tcm diagnosis standard: down in spirits, have a dull expression on one's face, lusterless complexion, hypoevolutism, mental incapacity.Slurred speech, active, absent minded, poor memory, school grade obviously falls behind, red tongue or red, few tongue or no fur, deep-thready pulse.
2) with reference to WHO1985 mental retardation standard revision standard:
(1) integration capability (language, memory, study, life, social adaptation) is lower than the child of the same age.
(2) morbidities in the past in 14 years old
(3) IQ index (IQ) is lower than 75.Mental retardation person due to the brain organic diseases such as autosomal abnormalities, innate immunity defective, various brain disorder sequela except needing.IQ is measured and is adopted Haskell Wexler-Liaoning 1986 revision scales.Respectively detect 1 time before and after the treatment, and judge with reference to other index comprehensives.
3. medicine and technology
Radix Polygoni Multiflori (system) 375g Semen Platycladi 175g Arillus Longan 125g
Rhizoma Polygonati 375g Fructus Ligustri Lucidi 375g Fructus Mori 375g
Semen Nelumbinis 175g Radix Polygalae 175g Poria 75g
Os Draconis 300g Bulbus Lilii 175g Radix Salviae Miltiorrhizae 250g
Ethyl hydroxybenzoate 0.1g white sugar 100g propylparaben 0.1g
Make 1000ml altogether.
[method for making] above 12 flavors add decocting, fry in shallow oil three times, add 7 times of amounts of water for the first time, flood 2 hours, decoct 1.5 hours, filter.For the second time add 5 times of amounts of water, decocted 1 hour, filter.Add 3 times of amounts of water for the third time, decocted 0.5 hour, filter.Merging filtrate leaves standstill more than 24 hours filtration under diminished pressure.Filtrate decompression is concentrated into no more than 925ml, adds white sugar ethyl hydroxybenzoate and propyl ester and stirs and to make dissolving, adds distilled water again to 1000ml, detects liquid PH value (being 4.5-5.5), after relative density (being 1.10-1.16) is qualified, and filtration under diminished pressure again.Fill, sterilization, promptly.
4. observational technique
The oral liquid of the oral medicine with intelligence increasing and brain tonic function of observation group is obeyed every day 2 times sooner or later, each every year old 1ml below 10 years old, and each 10ml more than 10 years old, 4 months is a course of treatment.Matched group adopts the intelligence training method.
5. efficacy determination
Produce effects: integration capability obviously improves, and IQ improves more than 15, and the traditional Chinese medical science is examined and seen that speech is clear, hypermnesia, and intelligence improves.
Take a turn for the better: integration capability increases, or certain intelligence (language, memory, understanding, operation) is obvious, and IQ improves more than 10; The traditional Chinese medical science is examined and is seen that speech is clearer, and memory before has enhancing, and intelligence increases.
Effectively: integration capability has raising, and IQ improves more than 5, and the traditional Chinese medical science is examined and seen that speech or memory have improvement, and intelligence slightly improves.
Invalid: every intelligent index slightly improves or no change.
6. observed result:
Total effective rate: observation group 82%, matched group 15% is learned by statistics and is handled P<0.005 difference highly significant, sees Table I for details.
Experimentation:
Adopt multiple model and method, inquired into the influence of the oral liquid of medicine with intelligence increasing and brain tonic function to animal learning memory, behavior, simultaneously from molecular level, subcellsular level has carried out comparatively deep research to its maincenter pharmacological mechanism.
Curative effect statistics before and after mental retardation observation group and the treatment of control group
Subordinate list 1
Total IQ (IQ) PIQ (IQ) linguistic IQ (IQ)
Treatment group treatment of control group group treatment of control group group matched group
The routine number % example of example number % number % example number % example number % example number % produce effects 13 26 11 11 17 111 11 11 9 10 11 11 improvements 12 24 11 11 111 211 11 11 12 21 11 11 effective 16 32 8 15 0 16 3 15 18 36 5 25 invalid 9 10 17 0.5 11 26 17 0.5 15 30 15 75 total effective 41 82 3 15 17 74 3 15 15 70 5 25XT, 31.5 22.4 14P<0.005 0.005 0.005
Materials and methods
(1) laboratory animal:
1. Kunming kind healthy mice, body weight 9-11g, the male and female dual-purpose, Chinese Medical Sciences University's Experimental Animal Center provides.
2.Wistar male white rat, body weight 150-200g, animal housing of Traditional Chinese Medicinal College of Liaoning provides.
(2) medicine and method
Medicine with intelligence increasing and brain tonic function is provided by the Hospital Attached to Liaoning Inst. of Traditional Chinese Medicine Drug Manufacturing Room.
1. learning and memory behavioral experiment
(1) medicine: piracetam is hated by herbal pharmacology teaching and research room of Shenyang Pharmacy College favour; Anisodine is hated by the institute of Materia Medica,Chinese Academy of Medical Sciences favour, and sodium nitrite and ethanol are the commercial reagent.
(2) experiment grouping: get 150 of mices, be divided into the blank group at random; Pathological model group (causing learning and memory to obtain, consolidate and reproduce the pathological model of obstacle for respectively Anisodine, sodium nitrite, 40% ethanol); The oral liquid I group of medicine with intelligence increasing and brain tonic function; The oral liquid II group of medicine with intelligence increasing and brain tonic function, the piracetam group.
(3) instrument: by imitated the forming of disposable training, avoidance conditioned reflex case of Zhang Juntian report.[1]
The diving tower reflective box is 20 * 20 * 30cm
3Wooden (-packing) case is the copper grid at the bottom of the case, passes to 40V electric current, case built-in one high 3.5cm, the valve rubber of diameter 5cm.The place of safety of avoiding electric shock as animal.
Keeping away dark reflective box is 40 * 15 * 15cm
3Glass box, divide light and shade two Room, the bottom, darkroom be the copper grid, passes to the 40V electric current, has the little edge of a diameter 3cm to communicate between two Room, bright chamber is that animal is avoided the place of safety of shocking by electricity.
(4) method: press document
[1]The report method.Blank group and pathological model group all gavage to normal saline before the experiment, meanwhile oral liquid I, the II of medicine with intelligence increasing and brain tonic function group and piracetam group gavage for respectively the oral liquid and the piracetam of the medicine with intelligence increasing and brain tonic function of various dose, secondary every day (early, evening respectively once).Continuous 10d.Train behind the last oral liquid of giving normal saline, medicine with intelligence increasing and brain tonic function and the piracetam, once training back 24h formally tests.Observation to learning and memory obtain influence the time, with each group mice 15min lumbar injection Anisodine 5mg/kg before training; Observation is consolidated when influence memory, subcutaneous injection sodium nitrite 120mg/kg immediately after training; When observation influenced memory represents, 10min gavaged 40% ethanol 0.1ml/10g body weight before the 24h test of training back.
(1) step down test: after during training mice being placed on reflective box endoadaptation 3min, energising 5min, record jumps off electric shock number of times (being errors number) from platform; Formally test behind 24h then, errors number in the record 3min is respectively as the observation index that memory is obtained, consolidates and reproduce.
(2) keep away dark test; Animal pierces in incubation period (pierce for the first time darkroom before time) in darkroom and the 5min and pierces the number of times in darkroom as observation index in the record test.
Above-mentioned two kinds of tests all can be observed the influence of the acquisition of oral liquid learning and memory, consolidation and the reproduction of medicine with intelligence increasing and brain tonic function by giving the learning and memory blocker of different links, all belong to the test of passive avoidance trained reflex.
Significance with bilateral " t " method of inspection analysis bank differences
2. brain acute anoxia experiment
(1) medicine: piracetam is so kind as to give by herbal pharmacology teaching and research room of Shenyang Pharmacy College; Sodium nitrite is Shenyang reagent one factory's product.
(2) grouping and administration: get 120 of mices, be divided into four groups at random, the normal saline group gavages to normal saline before the experiment, meanwhile oral liquid I, the II of medicine with intelligence increasing and brain tonic function group and piracetam group gavage oral liquid 12g crude drug/kg, the 36g crude drug/kg of medicine with intelligence increasing and brain tonic function respectively, piracetam 300mg/kg, secondary every day (early, evening each once) continuous 8 days, experimentizes behind the last administration 1b.
(3) method:
(1) broken end ischemia method:
[2]Cut mouse head, the mouth breathing with stopwatch record decapitated mice immediately continues time-histories.
(2) anoxia that causes of the sodium nitrite method that causes death:
[3]Give mouse subcutaneous injection sodium nitrite 225mg/kg once, record injection back is to the time of respiratory arrest.
(3) normobaric hypoxia method:
[4]Mice is put into the 250ml wide mouthed bottle that fills the 10g sodica calx, and bottleneck is coated with vaseline airtight, and the time-to-live of mice under the normobaric hypoxia state respectively organized in record.
Significance with bilateral " t " check analysis group difference.
3. focal cerebral ischemia rat brain born of the same parents Ultrastructural observation
Get 12 of rats, be divided into three groups at random, before the experiment, Chinese drug-treated group adopts free drinking technique to give the oral liquid 20g/kg of medicine with intelligence increasing and brain tonic function; Matched group and model group are given tap water, continuous 15 days with method.All use nembutal 40mg%kg intraperitoneal anesthesia during experiment.
(1) ligation right carotid
Behind the rat anesthesia, press document
[5,8]Method is isolated right carotid.Do not do ligation behind 4 rat surgical exposure of matched group common carotid artery; After model group and the Chinese drug-treated group surgical exposure right carotid, all do dual ligation, cut off for 4 every group at the ligation mid point.Broken end is got brain after 24 hours, gets cerebral cortex top fixation of tissue in 2.5% glutaraldehyde.
(2) pathologic finding:
Mus brain specimen starves through 1% that acid is fixing, ethanol acetone dewaters step by step, epoxy resin 618 embeddings again after 2.5% glutaraldehyde is fixing, ultrathin section, citron lead plumbate and the dyeing of acetic acid uranium, the saturating body electron microscopic observation of H-600.
4. monoamine neurotransmitters is measured in the mouse brain
(1) grouping: get 40 of mices, divide four groups at random, 10 every group, i.e. blank group, pathologic group, treatment group, Chinese drug-treated group.
(2) give approximately: the reserpine injection is
The state
Light restriction factory product.Blank group subcutaneous injection every day normal saline 0.1ml/10g gavages normal saline 0.2ml/10g simultaneously; Pathologic group subcutaneous injection of reserpine every day one physiology saline 0.25mg/kg, gavage normal saline 0.2ml/kg simultaneously, gavage the oral liquid 12%0.2ml/10g of medicine with intelligence increasing and brain tonic function simultaneously, Chinese drug-treated group subcutaneous injection every day normal saline 0.1ml/10g gavages the oral liquid 120%0.2ml/10g of medicine with intelligence increasing and brain tonic function simultaneously.Below respectively organize equal successive administration 8 days.
(3) method
High performance liquid chromatography-electrochemistry inspection hand hay cutter device (EPLC-ECD) monoamine neurotransmitter algoscopy.
(1) reagent
Methanol (AR), citric acid (A, R), di-n-butylamine (CR),
Sodium acetate (AR), EB
2EDTA
Shenyang reagent one factory's product
Perfluorooctane sulfonate, Sigeb company product
Norepinephrine (RE), dopamine (DA),
5-hydroxy tryptamine (5-HT), 5-hydroxyindoleacetic acid (5-DIAA)
3,4 dihydroxyphenyl acetic acids (DDPAC), dihydroxy benzylamine (DHBA)
Be Sigxa company and Kerck company product
Perchloric acid (AR) Shenyang reagent one factory's product
(2) instrument
The katers510 constant flow pump; 460 electrochemical detectors; Glass-carbon electrode; Resolve 5 μ Spherical cl6 chromatographic columns, 150 * 3.9mml/O; The U6k injector, 740 integrators.
(3) chromatographic condition
Flow rate pump 1.0ml/min, running voltage+0.70v, oxidizing process.Detection sensitivity 10nA; Chart speed 30cm/b; 20 ± 2 ℃ of room temperatures.
Mobile phase is formed: 50mmol/L sodium acetate, 20mmol/L citric acid, 1mmol/L perfluorooctane sulfonate, 1mmol/L di-n-butylamine, 0.2mmol/L, Rb
2EDTA, 2-4% methanol.With the preparation of deionization redistilled water, it is 4 that citric acid is transferred PH, and mixing filters ultrasonic degasification.
(4) standard substance and sample preparation
Standard substance preparation: standard substance NE, DA, 5-HT, 5-HIAA, DDFAC and interior mark DHBA are dissolved in 0.1mol/L, ECLD
4In the solution, be mixed with 10ng/ml concentration, put cryopreservation in the refrigerator, face with preceding and be diluted to a series of debita spissitudos with corresponding solvent.
Sample preparation: will get brain rapidly behind the mice broken end, decerebellation weigh and promptly put-40 ℃ of refrigerators freezing after, add 2mg/L DHBA50ml, cold 0.1mol/L perchloric acid 2ml makes homogenate (being cold operation), pour into then in the centrifuge tube in 4 ℃ 18000 rev/mins, centrifugal 30min gets supernatant 20 μ l direct injected.
(5) result calculates
The ratio of every kind of material peak height and DHBA peak height is made comparisons with the ratio in the standard substance in the sample, calculates the content of every gram wet tissue monoamine transmitters in the sample.Referring to Fig. 1,2.
Significance with bilateral " t " check analysis group difference
Result and analysis
(1) learning and memory behavioral experiment
1. the oral liquid of medicine with intelligence increasing and brain tonic function obtains bad to influence model group (giving Anisodine) and blank group relatively to learning and memory due to the Anisodine, and diving tower method experiment errors number obviously increases, and (P<0.01) is obviously shortened in the darkness avoidance test test incubation period; Oral liquid I, the II group and the model group of medicine with intelligence increasing and brain tonic function compare, diving tower method errors number obviously reduces (P<0.05, P<0.01), and II organizes darkness avoidance test significant prolongation incubation period (P<0.05), compare with model group, diving tower method experiment errors number significantly reduces (P<0.05).See Table 1.
2. the oral liquid of medicine with intelligence increasing and brain tonic function is consolidated bad influence to memory due to the sodium nitrite
Pathological model group (give sodium nitrite) and blank group relatively, diving tower experiment errors number significantly increases, and keeps away dark experiment and obviously shortens (P<0.01) incubation period; Oral liquid I, the II of medicine with intelligence increasing and brain tonic function group and pathological model group relatively, diving tower with keep away dark experiment errors number and all significantly reduce (P<0.01, P<0.05) test and obviously calculate long (P<0.01) incubation period.Piracetam and model group relatively, diving tower with keep away dark experiment errors number and also significantly reduce (P<0.05), long (P<0.05) is obviously calculated in test incubation period.See Table 2.
3. the oral liquid of medicine with intelligence increasing and brain tonic function is to the bad influence of memory represents due to 40% ethanol
Pathological model group (give 40% ethanol) and blank group relatively, diving tower experiment errors number obviously increases, darkness avoidance test obviously shortens (P<0.01) incubation period; Oral liquid I, the II group and the pathology matched group of medicine with intelligence increasing and brain tonic function compare, and diving tower experiment errors number significantly reduces (P<0.01), darkness avoidance test significant prolongation incubation period (P<0.05).Piracetam group and model group compare, and diving tower experiment errors number obviously reduces (P<0.05).See Table 3.
(2) oral liquid of medicine with intelligence increasing and brain tonic function is to the influence of period of development chmice acute cerebral anoxia state
In the broken end ischemia method, oral liquid I, the II group average mouth breathing time of mice of medicine with intelligence increasing and brain tonic function is all significantly calculated long (P<0.01) than the matched group time, sodium nitrite anoxia method, the oral liquid II group death time of piracetam and medicine with intelligence increasing and brain tonic function is all significantly calculated long (P<0.05, P<0.01) than matched group.In the experiment of normal pressure anoxia enduring, the oral liquid II group of piracetam group and medicine with intelligence increasing and brain tonic function all can prolong its life span (P<0.05, P<0.01).See Table 4.
(3) oral liquid of medicine with intelligence increasing and brain tonic function is to the Ultrastructural effect of focal rats with cerebral ischemia brain cell
1. matched group: mitochondrion quantity is more, and rounded or elongated shape, ridge are tabular or tubulose, and basic southern endoparticle is less.Endoplasmic reticulum often is close under the plasma membrane gathers multilamellar.Golgi complex is flourishing.(see figure 3).
2. model group: mitochondrial swelling, ridge disappear and are cavitation, and cellularity such as endoplasmic reticulum and Golgi complex are in various degree and change, and the cytoplasmic matrix electron density increases (seeing Fig. 4,5).
3. Chinese drug-treated group: the mitochondrion mild swelling, ridge exists, ridge quantity is bordering on normally, the cytoplasmic matrix electron density still can, other organelles do not have obvious change (seeing Fig. 6,7).
(4) oral liquid of medicine with intelligence increasing and brain tonic function is to the influence of monoamine neurotransmitter in the period of development mouse brain
1.RE concentration
Pathologic group (giving reserpine) compares with the blank group, and RE concentration significantly reduces (P<0.01): treatment group and pathologic group compare, and RE concentration obviously increases (P<0.01); Chinese drug-treated group and blank group compare, and RE concentration also increases (P<0.05).
2.DA, DDPAC concentration
Pathologic group (giving reserpine) compares with the blank group, and DA concentration significantly reduces.DDPAC concentration is 0 (P<0.01); Treatment group and pathologic group compare, and DA concentration obviously increases (P<0.01); Chinese drug-treated group and blank group compare, and DA concentration also increases (P<0.05).
3. 5-HT and 5-HIAA concentration pathologic group (giving reserpine) compare with the blank group, and 5-HT concentration significantly reduces (P<0.01), though 5-HIAA concentration descends, does not have significant difference; Treatment group and pathologic group compare, and 5-HT concentration obviously reduces (P<0.01), though 5-HIAA concentration reduces, difference does not have significance; Chinese drug-treated group and blank compare, no significant difference.
Conclusion:
(1) oral liquid of medicine with intelligence increasing and brain tonic function has facilitation to learning and memory function.
(2) the anti-acute brain anoxia functions of the oral liquid of medicine with intelligence increasing and brain tonic function and its memory improvement effect are closely related.
(3) oral liquid of medicine with intelligence increasing and brain tonic function may be that it promotes mechanism intelligent, that delay intellectual damage to the protective effect of cerebral cortex cell.
(4) oral liquid of medicine with intelligence increasing and brain tonic function promotes that the effect of learning and memory is relevant with monoamine neurotransmitter in the brain, and NE, DA concentration increase, and the 5-HT lowering of concentration may be the pharmacological Mechanism of its nootropic effect.
(2) treatment MBD80 example report (slight disordered brain function syndrome is called for short MBD)
1. clinical data
The MBD80 example all is institute of a section Pediatric Clinic case in the period of the 1988-1991,52 of men, and 26 of woman, divide observation group 50,30 of matched groups at random at 9.3 years old mean age.
2. diagnostic criteria
(1) the IQ index descends level (being lower than standard deviation of average) between two parties.
(2) understand, memory ability falls behind, hyperactivity, absent minded, learning difficulty (due to the various diseases except the person) is arranged in various degree.
3. medicine
The oral liquid of medicine with intelligence increasing and brain tonic function: Radix Polygoni Multiflori 325g, Semen Platycladi 150g, Arillus Longan 100g, Rhizoma Polygonati 325g, Fructus Ligustri Lucidi 325g, Fructus Mori 325g, Semen Nelumbinis 150g, Radix Polygalae 150g, Os Draconis 250g, Bulbus Lilii 150g, Radix Salviae Miltiorrhizae 225g, Poria 50g.
Technology: decocting-concentration method (providing) by the Drug Manufacturing Room of Traditional Chinese Medicinal College of Liaoning.
4. observational technique:
The oral liquid of the oral medicine with intelligence increasing and brain tonic function of observation group is obeyed every day 2 times sooner or later, each every year old 1ml below 10 years old, and each 10ml more than 10 years old, 4 months is a course of treatment.The oral piracetam of matched group, each 0.1, every day 3 times.
5. efficacy determination
Produce effects: school grade obviously improves, and hyperactivity obviously reduces, and the memory attention strengthens; The IQ index obviously improves.
Take a turn for the better: school grade, memory have certain amplitude to improve, and hyperactivity reduces, and the IQ index has raising.
Invalid: no change before and after the treatment.
6. therapeutic outcome:
Observation group's total effective rate 96%, matched group total effective rate 53% is learned by statistics and is handled P<0.05, difference highly significant.See Table II for details.
Table II MBD observation group and matched group coefficient ratio are
The matched group P of project observation group
Example (%) example (%)
Produce effects 24 (50) 5 (16)<0.005
Take a turn for the better 23 (46) 11 (39)
Invalid 2 (4) 14 (47)
Total effective 48 (96) 16 (53)
(3) treatment MR10 example report
1. clinical data
The MR10 example is Shenyang Inst. of Traditional Chinese Medicine's Pediatric Clinic case, male 8 examples, women 2 examples, 6.5 years old mean age.
2. diagnostic criteria.Same embodiment (one), the same embodiment of curative effect determinate standard ().
3. medicine and technology:
Radix Polygoni Multiflori 375g Semen Platycladi 175g Arillus Longan 100g Rhizoma Polygonati 350g
Fructus Ligustri Lucidi 375g Fructus Mori 325g Semen Nelumbinis 175g Radix Polygalae 175g
Technology: decocting-concentration method.
3. observational technique: the oral liquid of medicine with intelligence increasing and brain tonic function, each every year old 1ml, sooner or later oral every day 2 times, total 4 months courses of treatment.
Therapeutic outcome: produce effects 3 people, 3 people that take a turn for the better, effective 3 people, invalid 1 people.Total effective rate 90%.
(4) treatment neurasthenia, alzheimer disease, 30 example reports:
1. clinical data
My institute's internal medicine outpatient service is equipped with example, wherein male 18 examples, women 12 examples, 52 years old mean age in the period of all being equipped with examples and all being 1989-1992.
2. diagnosis and curative effect determinate standard are with reference to practical internal medicine standard.
3. medicine and technology
Radix Polygoni Multiflori 425g Semen Platycladi 200g Arillus Longan 150g Rhizoma Polygonati 425g
Fructus Ligustri Lucidi 425g Fructus Mori 425g Semen Nelumbinis 200g Radix Polygalae 200g
Poria 100g Os Draconis 350g Bulbus Lilii 200g Radix Salviae Miltiorrhizae 275g
Technology: decocting-concentration method is provided by the Drug Manufacturing Room of Traditional Chinese Medicinal College of Liaoning.
Observational technique: the oral liquid of medicine with intelligence increasing and brain tonic function, each 20ml obeys every day 2 times sooner or later, and 1-3 month is a course of treatment.
Therapeutic outcome: produce effects 12 examples (40%), 10 examples that take a turn for the better (33.3%), effective 6 examples (20%), invalid 2 examples (6.7%), total effective rate 93.3%.
The promoting intelligence and strengthening brain drug effect studies confirm that anti-acute brain anoxia functions, and the cerebral cortex cell is had protective effect, can delay the intelligence wound; so Yu Yonger adult neurasthenia, alzheimer disease, therapeutic outcome, total effective rate reaches 93.3%; illustrate that this oral liquid has slow down aging, the protection function of brain cell.
(5) anxious, slow toxicity test of the oral liquid of medicine with intelligence increasing and brain tonic function and medicine stability experiment:
1. anxious malicious conclusion (of pressure testing): irritate oral liquid 175g crude drug/kg that stomach gives the mice medicine with intelligence increasing and brain tonic function, be equivalent to 175 times of clinical child's consumption, mice does not have tangible poisoning manifestations, discloses the clinical a drug safety of this medicine.
2. long term toxicity test conclusion: continuous 12 weeks gavage oral liquid 19.6-79.2 (crude drug) 1kg of medicine with intelligence increasing and brain tonic function to rat oral, the 10-40 that is equivalent to child's one consumption per day more than 10 years old doubly, except that the high dose rat body weight increasess slowly, blood system, hepatic and renal function and pathologic finding etc. are not all had significantly influence, and this agent is taken in prompting does not for a long time have tangible accumulate poisoning reaction.
3. medicine stability test conclusion: the oral liquid of medicine with intelligence increasing and brain tonic function is through reserved sample observing 18 months, its character, and pH value, relative density, check result all meets the requirements.The discriminating item is checked out Radix Polygoni Multiflori, Fructus Mori.Health examination, assorted bacterium number, fungi count, yeast number average meet " ministry standard " version in 1986.Without going out escherichia coli, demodicid mite alive.Therefore, assert that this product is stable, qualified.