CN105055855B - With Chinese medicine composition for improving sleep, strengthen immunity effect and its preparation method and application - Google Patents
With Chinese medicine composition for improving sleep, strengthen immunity effect and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of Chinese medicine composition for having and improving sleep, strengthen immunity effect and its preparation method and application.It is provided by the present invention to be used to improve sleep, the Chinese medicine composition of strengthen immunity, it is made of wilsonii, American Ginseng, spina date seed, sealwort, psoralea corylifolia and Poria cocos.Through animal function test the results show that Chinese medicine composition provided by the present invention has the function that to improve sleep, strengthen immunity, animal safety result of the test shows, Chinese medicine composition safe without toxic side effect of the present invention, without teratogenesis mutagenesis.Human feeding trial shows that Chinese medicine composition of the present invention can effectively improve the symptoms such as insomnia, hypoimmunity, and significant effect, can be applied to the preparation of medicine or health products, have a vast market prospect.
Description
Technical field
The present invention relates to medicine or health products field of Chinese medicinal composition, and in particular to one kind, which has to improve, sleeps, strengthens and exempt from
Chinese medicine composition of epidemic disease power effect and preparation method and application.
Background technology
Sleep is a kind of spontaneous and reversible quiescent condition that higher vertebrate periodically occurs, and shows as body pair
The reactivity of environmental stimuli reduces and the temporary transient interruption of consciousness.The time in all one's life of people about 1/3 is spent in sleep.
When people are in sleep state, the brain and body of people can be made to be rested, rested and reorganized and recovered, suitable sleep has
Help the daily working and learning of people.Science improves sleep quality, is the guarantee that people work normally learning life.Sleep barrier
Hinder the exception of the exception for referring to amount of sleep and matter of sleeping, or some clinical symptoms occur in sleep, such as sleep reduces or sleep
Excessively, somnambulism etc..Difficulty falling asleep, it is shallow sleep easily wake up, wake up after have difficulty in going to sleep, dreaminess, be often accompanied by daytime is dispirited, four limbs without
The symptom such as power, slow in reacting, work efficiency is low, headache, failure of memory.Poor sleep and unpleasant pleasure of having a sleepless night, pay attention to
Power is not concentrated, and seriously affects daily life and work, and chronic insomnia person can increase the risk of the diseases such as hypertension and depression.
According to the relevant information, the people that the whole nation has more than 50% at present has difficulty in going to sleep and has a sleepless night phenomenon, due to long-term
Insomnia, many people have suffered from many relevant diseases.Sleep-disorder has become the big killer of the mankind, life, work to people
Make, study brings huge hidden danger.At present, all kinds of improvement sleep product of market prevalence is mainly sedative hypnotics and epiphysin
Class Chinese medicine composition.Current clinically common hypnotic has Benzodiazepines, barbiturates and other non-barbiturateses, it is controlled
It is obvious to treat the effect of insomnia, but has a great influence to its liver, kidney, user easily produces the dependence to medicine, usually not
The disconnected pharmaceutical dosage that increases could keep effect, but also can produce very big toxic side effect, therefore be not to be adapted to long-term use
Medicine.Epiphysin improves sleep effect and is still not clear, and long-term or high dose, which takes epiphysin, to produce harm to human body, such as
It is obvious to suppress human reproductive system's function.Person in middle and old age's (because melatonin secretion is reduced) are if crowd's epiphysin remains larger, meeting daytime
There is the dizzy and side effect such as shakiness on foot, danger may be brought compared with weak person to older, body.
Insomnia, the traditional Chinese medical science referred to as " being insomnia ", " must not sleep ", " must not crouch ", " insomnia " etc..The traditional Chinese medical science thinks that insomnia card has void
It is two kinds real, it is that body is interior caused by qi and blood, visceral dysfunction.Therefore, the treatment of insomnia, should focus on the medical treatment of internal organ, and
Qi and blood is set to reconcile, equilibrium between yin and yang, viscera function is attributed to normally.With reference to traditional Chinese medical theory, develop from safely, effectively improvement sleep
Chinese medicine composition it is imperative.
Immunity refers to the ability that human immune system resists pathogenic infection.Investigated according to the World Health Organization, the whole world is about
The people for having more than 35% is in fatigue state, and especially middle-aged male crowd fatigue state person is up to 60-75%.With life
The quickening of rhythm, people face each side such as family, cause, society surface pressure, often occur having a delicate constitution, is malnutritive, spirit
The hypoimmunities such as dispirited, fatigue and weak, appetite reduction, sleep-disorder show.Slight immunocompromised, passes through a period of time
Conditioning engine body can naturally recover, and serious immunocompromised not only needs the intervention of drug therapy, or even have and cause canceration
May.
The traditional Chinese medical science thinks the generation of disease, develops and lapses to the growth and decline depending on good and evil, related with the immune function of body, from
And propose the main principle of curing the disease of strengthening vital QI to eliminate pathogenic factors.Righting refers to help body healthy tendency, builds up health, to improve body anti-evil, anti-
A kind of principle of reatment of sick ability.Method by strengthening healthy tendency, eliminating evil are gone out, situation of getting well.Chinese medicine is in immunocompromised
Therapeutic effect protrude, can be controlled according to people's difference Syndrome Differentiation of Constitution opinion, give full play to immunity of organisms.
Studies have shown that immune system can obtain repairing to a certain degree during sleep, and transfer to make to having slept to adjust
With." healthy physique is from sleep " is the new viewpoint that medical researchers draw according to the latest result to sleep study.It is long
Phase sleep insufficiency can bring a series of body injury, including elaborative faculty decline, vigilance power and judgment to decline, and work(is immunized
Can low, endocrine disturbance etc..As it can be seen that there is very big contact between sleep quality and immunity.
In conclusion improve sleep and strengthen immunity has become the focus of growing interest, researching and developing one kind has
Sleep, strengthen immunity effect are can obviously improve, and the Chinese medicine composition having no toxic side effect has great importance.
The content of the invention
It is an object of the invention to provide a kind of Chinese medicine composition for having and improving sleep, strengthen immunity effect, it is adopted
It is made of pure plant, is had no toxic side effect to human body, can be long-term use of.Another object of the present invention is to provide the Chinese medicine group
The preparation method and applications of compound.
Technical solution, in order to realize the above object the technical solution that the present invention takes is：
It is a kind of have improve sleep, strengthen immunity effect Chinese medicine composition, it by following portions by weight bulk pharmaceutical chemicals
It is made：
2~10 parts of wilsonii, 1~10 part of American Ginseng, 5~30 parts of spina date seed, 5~20 parts of sealwort, 1~10 part of psoralea corylifolia,
5~30 parts of Poria cocos.
Preferably, the weight proportion of each bulk pharmaceutical chemicals is：
4~8 parts of wilsonii, 2~8 parts of American Ginseng, 10~20 parts of spina date seed, 6~16 parts of sealwort, 2~8 parts of psoralea corylifolia, Fu
10~20 parts of Siberian cocklebur.
The weight proportion of each bulk pharmaceutical chemicals is further preferably：
2 parts of wilsonii, 1 part of American Ginseng, 10 parts of spina date seed, 6 parts of sealwort, 3 parts of psoralea corylifolia, 10 parts of Poria cocos.
The weight proportion of each bulk pharmaceutical chemicals is further preferably：
4 parts of wilsonii, 2 parts of American Ginseng, 30 parts of spina date seed, 10 parts of sealwort, 8 parts of psoralea corylifolia, 30 parts of Poria cocos.
The weight proportion of each bulk pharmaceutical chemicals is further preferably：
4 parts of wilsonii, 8 parts of American Ginseng, 20 parts of spina date seed, 16 parts of sealwort, 2 parts of psoralea corylifolia, 10 parts of Poria cocos.
The weight proportion of each bulk pharmaceutical chemicals is further preferably：
6 parts of wilsonii, 6 parts of American Ginseng, 5 parts of spina date seed, 5 parts of sealwort, 7 parts of psoralea corylifolia, 5 parts of Poria cocos.
The weight proportion of each bulk pharmaceutical chemicals is further preferably：
8 parts of wilsonii, 2 parts of American Ginseng, 15 parts of spina date seed, 7 parts of sealwort, 3 parts of psoralea corylifolia, 20 parts of Poria cocos.
The weight proportion of each bulk pharmaceutical chemicals is further preferably：
6 parts of wilsonii, 5 parts of American Ginseng, 15 parts of spina date seed, 11 parts of sealwort, 5 parts of psoralea corylifolia, 15 parts of Poria cocos.
The weight proportion of each bulk pharmaceutical chemicals is further preferably：
5 parts of wilsonii, 5 parts of American Ginseng, 11 parts of spina date seed, 14 parts of sealwort, 3 parts of psoralea corylifolia, 18 parts of Poria cocos.
It is provided by the invention have improve sleep, the Chinese medicine composition of strengthen immunity effect, can by each bulk pharmaceutical chemicals and
Its extract and acceptable carrier are prepared into oral liquid, granule, capsule, tablet, pill.
The present invention based on Traditional Chinese medical theory, using wilsonii, American Ginseng, spina date seed, sealwort, psoralea corylifolia, Poria cocos as
Raw material.This product raw material is that 2010 editions pharmacopeia record kind, not in the row of " eighteen incompatibilities ", " nineteen counteraction ", no incompatibility.Thorn five
Add belong to tonic in Qi-tonifying drug, have strengthen the body resistance to consolidate the constitution, replenish qi to invigorate the spleen, soothing the liver kidney tonifying, intelligence promoting and tranquilization, strengthening the essence zhuanggu, promoting blood circulationization
The effect of the stasis of blood；American Ginseng is rare Chinese medicine, has boosting qi and nourishing yin, clearing heat and promoting fluid, adjust tonifying five zang organs, the effect for relieving restlessness of calming the nerves；Two
Person is combined into monarch drug in a prescription.Spina date seed is entered the warp of conscience, is had blood-nourishing tonifying liver, the effect of antitoxic heart-soothing and sedative with its sweet in flavor and neutral in nature；Sealwort has
Tonifying middle-Jiao and Qi, yin-nourishing, invigorating the spleen, moistening lung, the effect of kidney-nourishing；The two is ministerial drug；Psoralea corylifolia benefiting kidney-essence, warming kidney and enhancing yang；Poria cocos has
There are antitoxic heart-soothing and sedative, the effect of invigorating the spleen；The two is adjuvant.Each material combination of this product, complements each other, reach jointly improvement sleep,
The effect of strengthen immunity.
The preparation method of Chinese medicine composition of the present invention, it includes following processing steps：
A, in parts by weight, American Ginseng is taken, wilsonii, psoralea corylifolia, adds ethanol, heating and refluxing extraction, obtains ethanol extraction
Liquid；
B, the dregs of a decoction after the extraction of step A ethanol are taken to add water with spina date seed, sealwort, Poria cocos, heating extraction, obtains water extraction
Liquid；
C, step A ethanol extracts are concentrated into no ethanol flavor, the concentration of step B Aqueous extracts, merges to obtain concentrate；
D, preparation is made in concentrate obtained by step C and pharmaceutically acceptable carrier.
Preferably, the preparation of the above-described Chinese medicine composition that there is improvement sleep, strengthen immunity to act on
Method, it is characterised in that comprise the following steps：
A, American Ginseng is taken in parts by weight, and wilsonii, psoralea corylifolia, adds the 10 of the volume of 1~10 times of amount of medicinal material weight
~90% ethanol (volumetric concentration is), heating and refluxing extraction 1~3 time, every time 1~3 it is small when, merge to obtain alcohol extract；Ethanol returns
Receive；
B, spina date seed, sealwort, the Poria cocos of the dregs of a decoction and parts by weight after the extraction of step A ethanol are taken, adds 1~15 medicinal material times
The water of amount, heating extraction 1-3 times, every time 1~3 it is small when, merge to obtain aqueous extract；
C, step A alcohol extracts are concentrated into medicinal material equivalent as 0.8:1~2:1, step B Aqueous extracts are concentrated into medicinal material equivalent about
For 0.8:1~2:1, mixing, it is about 1 to continue to be concentrated into medicinal material equivalent:1~2:1；
D, by concentrate obtained by step C and the one or more in chitosan, xylitol, Sucralose, dextrin and starch
Combination, is made oral liquid, granule, capsule, tablet or pill.
As more preferably scheme, the system of the above-described Chinese medicine composition that there is improvement sleep, strengthen immunity to act on
Preparation Method, it comprises the following steps：
A：American Ginseng, wilsonii, psoralea corylifolia, the volumetric concentration of addition medicinal material 5 times of volumes of gross weight are taken in parts by weight
For 50% ethanol, heating and refluxing extraction 2 times, every time 2 it is small when, merge to obtain alcohol extract；
B：The alcohol extracting dregs of a decoction add the water of 10 times of amounts with spina date seed, sealwort, Poria cocos, and heating decocts extraction 2 times, and 2 is small every time
When, merge to obtain aqueous extract；
C：Alcohol extract is concentrated into no ethanol flavor, medicinal material equivalent is about 1 (with respect to alcohol extracting part):1, Aqueous extracts are concentrated into medicine
Material equivalent is about 1:1, mixed with alcohol extracting concentrate, it is about 1.5 to continue to be concentrated into medicinal material equivalent:1.
Beneficial effect：It is provided by the invention that there is improvement sleep, the Chinese medicine composition of strengthen immunity effect and its preparation
Compared to the prior art method has the following advantages：
The theory that the present invention is controlled according to Chinese herb decoction opinion, the Chinese medicine of optimal prescription proportioning is preferably obtained by many experiments
Composition, Chinese medicine composition medication is rigorous, and proportioning is scientific and reasonable.
The present invention is filtered out optimal extraction separation method by many experiments, extracted according to the physicochemical property of each flavour of a drug
Rational technology, workable, the active constituent content extracted is high, and impurity is few.
Chinese medicine composition provided by the invention warp the Pharmacological experiment result shows that, can obviously improve sleep and strengthen immunity effect
Fruit, and it is long-term use of, have no toxic side effect.
Improve sleep function experiment：With Chinese medicine composition oral liquid 0.5ml/kg/d, 1.0ml/kg/d, 3.0ml/ of the present invention
Tri- dosage groups of kg/d give mouse one month, carry out direct sleep experiments, the extension yellow Jackets length of one's sleep tests, penta bar
Tested than appropriate sodium sub-threshold dose sleep experiments, barbital sodium Sleep latency.The result shows that Chinese medicine composition oral liquid of the present invention
0.5ml/kg/d, 1.0ml/kg/d, 3.0ml/kg/d group act on mouse without direct sleep.3.0ml/kg/d group mice sleeps
Time is longer than 0ml/kg/d groups, and mice sleep incubation period is significantly shorter than 0ml/kg/d groups, has with 0ml/kg/d groups comparing difference
Statistical significance (P ＜ 0.05).According to《Health food is examined and assessment technique specification》Improvement sleep function experiment result
Judge, Chinese medicine composition of the present invention has improvement sleep function.
Strengthen immunity function test：With Chinese medicine composition oral liquid 0.5ml/kg/d, 1.0ml/kg/d of the present invention,
The dosage group orally administration mouse one month of 3.0ml/kg/d tri-, carry out mouse cell immune function, humoral immune function, list
Core-macrophage function and NK cytoactive detections, the results show 0.5ml/kg/d, 1.0ml/kg/d, 3.0ml/kg/d group add
ConA holes and the difference for being not added with ConA holes absorbance, the weightening of 3.0ml/kg/d groups auricular concha are above 0ml/kg/d groups；3.0ml/kg/
D groups half hemolytic value, hemolysis plaque number are higher than 0ml/kg/d groups；3.0ml/kg/d group NK cytoactives are higher than 0ml/kg/d groups,
And difference has statistical significance (P ＜ 0.05).According to《Health food is examined and assessment technique specification》Strengthen immunity work(
The result judgement that can be tested, Chinese medicine composition of the present invention have the function of strengthen immunity.
Acute oral toxicity test and 30 days feeding trials：Chinese medicine composition provided by the invention is female, male mice is anxious
The oral MTD of property is more than 22400mg/kg, and according to acute toxicity dose grading standard, Chinese medicine composition of the present invention belongs to nontoxic level.
Chinese medicine composition of the present invention (recommends agent with 3.0mg/kg/d, 6.5mg/kg/d, 10.0mg/kg/d equivalent to human body
Amount 30 times, 65 times, 100 times) dosage give rat 30 days, animal subject ordinary circumstance is good, weight, food utilization,
The change without exception of organ weights, organ coefficient；Hematological indices and biochemical indicator the results show that indices in normal model
Enclose；Each internal organs histopathologic examination is showed no the pathological change related with given the test agent, according to the Ministry of Public Health《Health food is examined
With assessment technique specification》30 days feeding trials of (version in 2003), give the Chinese medicine of the present invention of 100 times of animal human body recommended dose
Composition, does not observe illeffects.
Teratogenesis mutagenicity test：Test result indicates that each dosage group Salmonella reversion test of Chinese medicine composition of the present invention, mouse bone marrow cells
Cell micronucleus test and mouse inbred strain result are feminine gender, illustrate Chinese medicine composition of the present invention without teratogenesis mutagenesis
Effect, securely and reliably.
The present invention, which has also carried out Chinese medicine composition, improves sleep and strengthen immunity human feeding trial, improves sleep laboratory
Itself compare and (P ＜ are obviously reduced in control group comparison among groups, clinical symptoms integration the result shows that test-meal group test-meal is front and rear
0.01), test-meal group crowd sleep quality is obviously improved, and does not report other side effects, and Chinese medicine composition of the present invention has improvement to sleep
The function of dormancy.Strengthen immunity result of the test shows that the content of lymphocyte and macrophage substantially increases before and after test-meal group test-meal
Add (P ＜ 0.01), hence it is evident that higher than control group, two comparison among groupss have significant difference (P ＜ 0.01), illustrate the invention Chinese traditional medicine composition
Thing has the function of enhancing immunity of organisms.
Embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
Apply among a scope.After the present invention has been read, modification of the those skilled in the art to the various unsubstantialities of the present invention is equal
Fall within the application appended claims limited range.
Embodiment 1
Chinese medicine composition oral liquid of the present invention
1st, raw material weight number
2 parts of wilsonii, 1 part of American Ginseng, 10 parts of spina date seed, 6 parts of sealwort, 3 parts of psoralea corylifolia, 10 parts of Poria cocos.
2nd, preparation process
(1) American Ginseng is taken, wilsonii, psoralea corylifolia, adds 5 times of 50% ethanol (volumetric concentration) measured of medicinal material weight,
Heating and refluxing extraction 2 times, every time 2 it is small when, merge to obtain alcohol extract；Ethanol recycles；
(2) step (1) ethanol extraction dregs of a decoction are taken to add the water of 10 times of amounts with spina date seed, sealwort, Poria cocos, heating decocts extraction
2 times, every time 2 it is small when, merge to obtain aqueous extract；
(3) alcohol extract is concentrated into no ethanol flavor, medicinal material equivalent is about 1 (with respect to alcohol extracting part):1, Aqueous extracts are concentrated into
Medicinal material equivalent is about 0.8:1, mixed with alcohol extracting concentrate, it is about 1 to continue to be concentrated into medicinal material equivalent:1.
(4) chitosan is made into 1% aqueous solution, medicine liquid heating adds chitosan liquid while stirring to 60 DEG C, stirs 30 points
Clock, centrifugation, obtains clarified solution.
(5) xylitol, Sucralose are dissolved with purified water, added in clarified solution, purified water is settled to amount of preparation, adjusts PH
To 6.When 0-5 DEG C of refrigeration standing 24 is small, 0.45 μm of filtering with microporous membrane, obtains oral liquid of the present invention.
Embodiment 2
Chinese medicinal composition granules of the present invention
1st, raw material weight number
4 parts of wilsonii, 2 parts of American Ginseng, 30 parts of spina date seed, 10 parts of sealwort, 8 parts of psoralea corylifolia, 30 parts of Poria cocos.
2nd, preparation process
(1) above-mentioned American Ginseng is taken, wilsonii, psoralea corylifolia, adds 60% ethanol of 8 times of amounts of medicinal material weight, be heated to reflux carrying
Take 2 times, every time 1 it is small when, merge to obtain alcohol extract；Ethanol recycles；
(2) step (1) ethanol extraction dregs of a decoction are taken to add the water of 8 times of amounts with spina date seed, sealwort, Poria cocos, heating decocts extraction 2
It is secondary, every time 1 it is small when, merge to obtain aqueous extract；
(3) alcohol extract is concentrated into no ethanol flavor, medicinal material equivalent is about 0.8 (with respect to alcohol extracting part):1, Aqueous extracts concentration
It is about 0.8 to medicinal material equivalent:1, mixed with alcohol extracting concentrate, it is about 1 to continue to be concentrated into medicinal material equivalent:1.
(4) concentrate is added into dextrin, starch, be uniformly mixed, whole grain, it is dry, granule is made.
Embodiment 3
Chinese medicinal composition capsules agent of the present invention
1st, raw material weight number
4 parts of wilsonii, 8 parts of American Ginseng, 20 parts of spina date seed, 16 parts of sealwort, 2 parts of psoralea corylifolia, 10 parts of Poria cocos.
2nd, preparation process
(1) above-mentioned American Ginseng is taken, wilsonii, psoralea corylifolia, adds 80% ethanol of total 10 times of amounts of medicinal material weight, be heated to reflux
Extraction 2 times, every time 2 it is small when, merge to obtain alcohol extract；Ethanol recycles；
(2) extraction of step (1) ethanol dregs of a decoction and spina date seed, sealwort, Poria cocos is taken to add the water of 5 times of amounts, heating extraction 2 times,
Every time 2 it is small when, merge to obtain aqueous extract；
(3) alcohol extract is concentrated into no ethanol flavor, medicinal material equivalent is about 1 (with respect to alcohol extracting part):1, Aqueous extracts are concentrated into
Medicinal material equivalent is about 1:1, mixed with alcohol extracting concentrate, it is about 2 to continue to be concentrated into medicinal material equivalent:1.
(4) above-mentioned concentrate and starch, dextrin are mixed, paste block is made, it is dry, add starch appropriate, be ground into fine powder, mistake
Sieve, mixes, encapsulated with the general pelletization of water.
Embodiment 4
Chinese medicine composition tablet of the present invention
1st, raw material weight number
6 parts of wilsonii, 6 parts of American Ginseng, 5 parts of spina date seed, 5 parts of sealwort, 7 parts of psoralea corylifolia, 5 parts of Poria cocos.
2nd, preparation process
(1) above-mentioned American Ginseng is taken, wilsonii, psoralea corylifolia, adds 70% ethanol of medicinal material 3 times of volumes of weight, heat back
Stream extraction 3 times, every time 1 it is small when, merge to obtain alcohol extract；Ethanol recycles；
(2) extraction of step (1) ethanol dregs of a decoction and spina date seed, sealwort, Poria cocos is taken to add the water of 6 times of amounts, heating extraction 3 times,
Every time 1 it is small when, merge to obtain aqueous extract；
(3) alcohol extract is concentrated into no ethanol flavor, medicinal material equivalent is about 2 (with respect to alcohol extracting part):1, Aqueous extracts are concentrated into
Medicinal material equivalent is about 1:1, mixed with alcohol extracting concentrate, it is about 1.5 to continue to be concentrated into medicinal material equivalent:1.
(4) concentrate is added into dextrin, starch, be uniformly mixed, whole grain, dry, tabletting, is made tablet.
Embodiment 5
Chinese medicine composition pill of the present invention
1st, raw material weight number
8 parts of wilsonii, 2 parts of American Ginseng, 15 parts of spina date seed, 7 parts of sealwort, 3 parts of psoralea corylifolia, 20 parts of Poria cocos.
2nd, preparation process
(1) above-mentioned American Ginseng is taken, wilsonii, psoralea corylifolia, adds 90% ethanol of medicinal material 3 times of volumes of weight, heat back
Stream extraction 3 times, every time 1 it is small when, merge to obtain alcohol extract；Ethanol recycles；
(2) step (1) ethanol extraction dregs of a decoction are taken to add the water of medicinal material 6 times of volumes of weight with spina date seed, sealwort, Poria cocos,
Heating decoct extraction 3 times, every time 1 it is small when, merge to obtain aqueous extract；
(3) step (1) ethanol extract is concentrated into no ethanol flavor, medicinal material equivalent is about 2 (with respect to alcohol extracting part):1, water carries
It is about 1.5 that liquid, which is concentrated into medicinal material equivalent,:1, mixed with alcohol extracting concentrate, it is about 2 to continue to be concentrated into medicinal material equivalent:1.
(4) concentrate added into appropriate dextrin, be uniformly mixed, it is dry, closed and sticked together with 85% ethanol, ball is made in modeling, dry, activity
Charcoal is coated to obtain the final product.
Embodiment 6
Chinese medicine composition oral liquid of the present invention
1st, raw material weight number
6 parts of wilsonii, 5 parts of American Ginseng, 15 parts of spina date seed, 11 parts of sealwort, 5 parts of psoralea corylifolia, 15 parts of Poria cocos.
2nd, preparation process
(1) American Ginseng is taken, wilsonii, psoralea corylifolia, adds 7 times of 50% ethanol (volumetric concentration) measured of medicinal material weight,
Heating and refluxing extraction 2 times, every time 1.5 it is small when, merge to obtain alcohol extract；Ethanol recycles；
(2) step (1) ethanol extraction dregs of a decoction are taken to add the water of 12 times of amounts with spina date seed, sealwort, Poria cocos, heating decocts extraction
2 times, every time 1.5 it is small when, merge to obtain aqueous extract；
(3) alcohol extract is concentrated into no ethanol flavor, medicinal material equivalent is about 1.5 (with respect to alcohol extracting part):1, Aqueous extracts concentration
It is about 2 to medicinal material equivalent:1, mixed with alcohol extracting concentrate, it is about 2 to continue to be concentrated into medicinal material equivalent:1.
(4) chitosan is made into 1% aqueous solution, medicine liquid heating adds chitosan liquid while stirring to 60 DEG C, stirs 30 points
Clock, centrifugation, obtains clarified solution.
(5) xylitol, Sucralose are dissolved with purified water, added in clarified solution, purified water is settled to amount of preparation, adjusts PH
To 5.When 0-5 DEG C of refrigeration standing 24 is small, 0.45 μm of filtering with microporous membrane, obtains oral liquid of the present invention.
Embodiment 7
Chinese medicinal composition granules of the present invention
1st, raw material weight number
5 parts of wilsonii, 5 parts of American Ginseng, 11 parts of spina date seed, 14 parts of sealwort, 3 parts of psoralea corylifolia, 18 parts of Poria cocos.
2nd, preparation process
(1) above-mentioned American Ginseng is taken, wilsonii, psoralea corylifolia, adds 60% ethanol of 5 times of amounts of medicinal material weight, be heated to reflux carrying
Take 2 times, every time 2 it is small when, merge to obtain alcohol extract；Ethanol recycles；
(2) step (1) ethanol extraction dregs of a decoction are taken to add the water of 14 times of amounts with spina date seed, sealwort, Poria cocos, heating decocts extraction
2 times, every time 1 it is small when, merge to obtain aqueous extract；
(3) alcohol extract is concentrated into no ethanol flavor, medicinal material equivalent is about 1 (with respect to alcohol extracting part):1, Aqueous extracts are concentrated into
Medicinal material equivalent is about 1:1, mixed with alcohol extracting concentrate, it is about 1.5 to continue to be concentrated into medicinal material equivalent:1.
(4) concentrate is added into dextrin, starch, be uniformly mixed, whole grain, it is dry, granule is made.
Effect example 1
Chinese medicine composition of the present invention improves sleep function experiment
1 materials and methods
1.1 sample：The oral liquid that the embodiment of the present invention 1 is prepared, specification：30ml/ branch, lot number：20141001 samples
Form is brown color liquid, puts and is preserved at shady and cool dry and ventilated.
1.2 experimental animal：Select the SPF levels CI/F1 generations of the western Poole-Bi Kai experimental animals Co., Ltd breeding in Shanghai strong
Health female mice 120, weight 19.0-21.9g, experimental animal production licence number are SCXK (Shanghai) 2013-0016, the quality certification
Number：2008001643010.
Mouse adapts to environment (temperature is 20-25 DEG C, relative humidity 40-70%), and quarantine is randomly divided into after 4 days by weight
Three big group, wherein I group of mouse 40, is randomly divided into 4 dosage groups, every group 10, first carries out direct sleep experiments, next day is again
Carry out extending the experiment of the yellow Jackets length of one's sleep；II group of mouse 40, is randomly divided into 4 dosage groups, every group 10, carries out penta
The hypnosis of barbital sodium sub-threshold dose is tested；III group of mouse 40, is randomly divided into 4 dosage groups, every group 10, carries out barbital sodium
Sleep latency is tested.
1.3 dosage select to give mode with tested material：If tri- agent of 0.5ml/kg/d, 1.0ml/kg/d, 3.0ml/kg/d
Amount group (5 times, 10 times, 30 times that are respectively equivalent to given the test agent human body recommended intake), separately sets 0ml/kg/d groups with sterile water
Instead of tested material.Given the test agent is prepared with sterile water, and basic, normal, high dosage formulation concentration is respectively 5%, 10%, 30%, orally
The tested material of mouse corresponding dosage is given once a day, and intragastric administration on mice dosage is 0.1ml/10g.
1.4 key instruments and reagent
1.4.1 key instrument：T1000 types electronic balance, JA2003 types electronic balance, electronic stopclock.
1.4.2 main agents：Yellow Jackets, barbital sodium.
1.5 test method
According to《Health food is examined and assessment technique specification》The improvement sleep function method of inspection of (version in 2003) carries out.
1.5.1 the direct sleep experiments of mouse
Each dosage group animal continuous gavage one month, after the last administration, using mouse righting reflex loss 1min to enter
Sleep, righting reflex recover to be mouse awakening criterion, record each group sleep number of animals and the length of one's sleep.
1.5.2 the experiment of the yellow Jackets length of one's sleep is extended
Each dosage group animal continuous gavage one month, after last dose 15min, each group animal presses 40mg/kg dosage abdomens
Chamber injects yellow Jackets, and injection volume 10mg/kg, rights disappearance 1min as sleep index using mouse, observe given the test agent energy
The no extension yellow Jackets length of one's sleep.Compare the difference between control group and the extension of the experimental group length of one's sleep, the length of one's sleep prolongs
With conspicuousness, then experimental result is positive.
1.5.3 the hypnosis of yellow Jackets sub-threshold dose is tested
Each dosage group animal continuous gavage one month, after last dose 15min, each group animal presses 35mg/kg dosage abdomens
Chamber injects yellow Jackets, and injection volume 10ml/kg, rights disappearance 1min as sleep index using mouse, record to amobarbital
Each group sleep number of animals in sodium 30min.Compare the difference between control group and experimental group sleep number of animals, incidence of sleep animals
Increase has conspicuousness, then experimental result is positive.
1.5.4 barbital sodium Sleep latency is tested
Each dosage group animal continuous gavage one month, after last dose 15min, each group animal presses 300mg/kg dosage abdomens
Chamber inject barbital sodium, injection volume 10ml/kg, using mouse right disappearance 1min for sleep index, observe given the test agent to bar
Than the influence of appropriate sodium Sleep latency.Compare the difference between control group and experimental group Sleep latency, Sleep latency shortens
There is conspicuousness, then experimental result is positive.
1.6 test datas count：Homogeneity test of variance is carried out to each experiment initial data with SPSS16.0 softwares, is met
The data information that variance requires together, with mean between multiple test groups in one-way analysis of variance method and a control group two-by-two
Comparative approach carries out statistical disposition；Appropriate variable conversion is carried out to the data information of Non-Gaussian Distribution or heterogeneity of variance, is treated full
After sufficient normal state or variance require together, statistical disposition is carried out with the data of conversion gained.
2 results
Drinking water for animals is ingested normally in experimentation, and appearance is without exception.
The influence of 2.1 Chinese medicine composition Oral Liquid On Mice weight of the present invention
By table 1-1 to table 1-3 as it can be seen that original body mass 0.5ml/kg/d, 1.0ml/kg/d, 3.0ml/kg/d group of mouse with
0ml/kg/d groups compare, and there are no significant for difference (P ＞ 0.05).Show that the original body mass of mouse is more balanced between each group.
The Chinese medicine composition oral liquid of the present invention of orally administration mouse various dose one month, each dosage group weight and weight
Increase weight through homogeneity test of variance, meet homogeneity of variance requirement, it is right with multiple experimental groups in one-way analysis of variance method and one
Statistical disposition is carried out according to the comparative approach two-by-two of mean between group.0.5ml/kg/d, 1.0ml/kg/d, 3.0ml/kg/d group and 0ml/
Kg/d compares, and difference is not statistically significant (P ＞ 0.05).
Influence of the table 1-1 Chinese medicine composition oral liquids of the present invention to I group of mouse weight
Influence of the table 1-2 Chinese medicine composition oral liquids of the present invention to II group of mouse weight
Influence of the table 1-3 Chinese medicine composition oral liquids of the present invention to III group of mouse weight
The influence that 2.2 Chinese medicine composition Oral Liquid On Mices of the present invention are directly slept
The Chinese medicine composition oral liquid of the present invention of orally administration mouse various dose one month, after the last administration, each group is small
Phenomenon of sleeping does not occur for mouse.By table 2 as it can be seen that Chinese medicine composition oral liquid 0.5ml/kg/d, 1.0ml/kg/d of the present invention,
3.0ml/kg/d groups act on mouse without direct sleep.
The influence that the Chinese medicine composition Oral Liquid On Mice of the present invention of table 2 is directly slept
Influence of the 2.3 Chinese medicine composition oral liquids of the present invention to the extension yellow Jackets inducing mouse length of one's sleep
The Chinese medicine composition oral liquid of the present invention of orally administration mouse various dose one month, is injected intraperitoneally amobarbital
Sodium, observes the length of one's sleep of mouse.By 3 result of table as it can be seen that 3.0ml/kg/d group mouse sleep times are considerably longer than 0ml/kg/d
Group (P ＜ 0.05).
Influence of the Chinese medicine composition oral liquid of the present invention of table 3 to the extension yellow Jackets inducing mouse length of one's sleep
Influence of the 2.4 Chinese medicine composition oral liquids of the present invention to yellow Jackets sub-threshold dose hypnosis
The Chinese medicine composition oral liquid of the present invention of orally administration mouse various dose one month, is injected intraperitoneally amobarbital
Sodium, observes each group mice sleep incidence in 30min.By 4 result of table as it can be seen that 0.5ml/kg/d, 1.0ml/kg/d, 3.0ml/
Compared with 0ml/kg/d groups, 3.0ml/kg/d group animal sleep incidences increase kg/d group animal sleep incidences.
Shadow of the Chinese medicine composition oral liquid of the present invention of table 4 to yellow Jackets sub-threshold dose inducing mouse sleep incidence
Ring
Influence of the 2.5 Chinese medicine composition oral liquids of the present invention to barbital sodium Sleep latency
The Chinese medicine composition oral liquid of the present invention of orally administration mouse various dose one month, is injected intraperitoneally barbital sodium,
Observe the Sleep latency of mouse.By table 5 as it can be seen that 3.0ml/kg/d group mice sleep incubation periods are significantly shorter than 0ml/kg/d groups (P
＜ 0.05).
Influence of the Chinese medicine composition oral liquid of the present invention of table 5 to barbital sodium Sleep latency
According to《Health food is examined and assessment technique specification》Improvement sleep function experiment result judgement, the present invention in
Drug composition has improvement sleep function.
Effect example 2
Chinese medicine composition strengthen immunity function test of the present invention
1 materials and methods
1.1 sample：1 gained oral liquid of the embodiment of the present invention.Sample morphology is brown color liquid, puts shady and cool dry and ventilated
Place preserves.
1.2 experimental animal：Select the SPF levels CI/F1 generations of the western Poole-Bi Kai experimental animals Co., Ltd breeding in Shanghai strong
Health female mice 200, weight 18.6-21.3g, experimental animal production licence number are SCXK (Shanghai) 2013-0016, the quality certification
Number：2008001644438.
Mouse is randomly divided into I, II, III, IV, V 5 big group by weight, per 40 mouse of group greatly, is divided into 4 dosage groups,
Each dosage group 10.Wherein I group of mouse carries out the mouse spleen lymphocyte conversion of ConA inductions, NK cell activity assays；Ⅱ
Group mouse carries out auricle edema experiment；III group of mouse carries out antibody-producting cell detection and half hemolytic value HC50Measure；IV group
Mouse carries out mouse carbonic clearance test；V group of mouse carries out Turnover of Mouse Peritoneal Macrophages phagocytosis chicken red blood cell experiment.Raising temperature
For 20-25 DEG C, relative humidity 40-70%
1.3 dosage select to give mode with tested material：If tri- agent of 0.5ml/kg/d, 1.0ml/kg/d, 3.0ml/kg/d
Amount group (5 times, 10 times, 30 times that are respectively equivalent to given the test agent human body recommended intake), separately sets 0ml/kg/d groups with sterile water
Instead of tested material.Given the test agent is prepared with sterile water, and basic, normal, high dosage formulation concentration is respectively 5%, 10%, 30%, orally
The tested material of mouse corresponding dosage is given once a day, and intragastric administration on mice dosage is 0.1ml/10g.Continuous gavage measures after one month
Every strengthen immunity functional parameter.
1.4 key instruments and reagent
1.4.1 key instrument：Card punch, T1000 types electronic balance, JA2003 types electronic balance, SG-603 bio-safeties
Cabinet, multi-function microplate reader, CO2gas incubator, JJ-100 electronic balances.
1.4.2 main agents：DNFB, SRBC, guinea pig serum, india ink, RPMI1640, ConA, MTT, isopropanol,
SA buffer solutions, Dou Shi reagents, YAC-1 cells, LDH matrix liquids.
1.5 test method：According to《Health food is examined and assessment technique specification》The strengthen immunity work(of (version in 2003)
Can method of inspection progress.1. ConA inducing mouses Splenic vein hemodynamics experiment-mtt assay is carried out respectively；2. DNFB inducing mouses
Delayed allergy (DTH)-ear swelling method；3. antibody-producting cell detection-Jerne improves slide methods；4. serum hemolysin
Measure-half hemolytic value (HC50)；5. mouse carbonic clearance test；6. Turnover of Mouse Peritoneal Macrophages phagocytosis chicken red blood cell experiment-halfbody
Interior method；7. NK cytoactive detections-determination of lactate dehydrogenase method.
1.6 test datas count：Homogeneity test of variance is carried out to each experiment initial data with SPSS16.0 softwares, is met
The data information that variance requires together, with mean between multiple test groups in one-way analysis of variance method and a control group two-by-two
Comparative approach carries out statistical disposition；Appropriate variable conversion is carried out to the data information of Non-Gaussian Distribution or heterogeneity of variance, is treated full
After sufficient normal state or variance require together, statistical disposition is carried out with the data of conversion gained.
1.7 result judgement：According to《Health food is examined and assessment technique specification》, in cellular immune function, (ConA is induced
Mouse spleen lymphocyte conversion, DNFB inducing mouse DTH), humoral immune function (antibody-producting cell detection, mouse peritoneal
Macrophage swallows chicken red blood cell), any two aspect results of the aspect of NK cytoactives four it is positive, can determine that the given the test agent has
There is strengthen immunity function.
Two result of the tests wherein in cellular immune function assay project are the positive, or two agent of any one experiment
Amount group result is positive, can determine that the cellular immune function assay result positive.Two experiment knots in humoral immune function measurement result
Fruit is the positive, or two dosage group results of any one experiment are positive, can determine that the humoral immune function measurement result positive.It is single
Two result of the tests are the positive in core macrophage function measurement result, or two dosage group results sun of any one experiment
Property, it can determine that the mononuclear macrophage functional examination result positive.More than one dosage group result of NK cytoactive detections experiment
The positive, can determine that the NK cytoactives result positive.
2 results
Drinking water for animals is ingested normally in experimentation, and appearance is without exception.
The influence of 2.1 Chinese medicine composition Oral Liquid On Mice weight of the present invention
By table 6-1 to table 6-5 as it can be seen that original body mass 0.5ml/kg/d, 1.0ml/kg/d, 3.0ml/kg/d group of mouse with
0ml/kg/d groups compare, no significant difference (P ＞ 0.05).Show that the original body mass of mouse is more balanced between each group.
The Chinese medicine composition oral liquid of the present invention of orally administration mouse various dose one month, by each dosage group weight and body
Weightening value meets homogeneity of variance requirement, with multiple test groups and one in one-way analysis of variance method through homogeneity test of variance again
The comparative approach two-by-two of mean carries out statistical disposition between a control group.0.5ml/kg/d, 1.0ml/kg/d, 3.0ml/kg/d group with
0ml/kg/d compares, no significant difference (P ＞ 0.05).
The influence of table 6-1 Chinese medicine composition Oral Liquid On Mice weight of the present invention
The influence of table 6-2 Chinese medicine composition Oral Liquid On Mice weight of the present invention
The influence of table 6-3 Chinese medicine composition Oral Liquid On Mice weight of the present invention
The influence of table 6-4 Chinese medicine composition Oral Liquid On Mice weight of the present invention
The influence of table 6-5 Chinese medicine composition Oral Liquid On Mice weight of the present invention
The influence of 2.2 Chinese medicine composition Oral Liquid On Mice thymus gland, spleen organ of the present invention
The 1 Chinese medicine composition oral liquid of the embodiment of the present invention of orally administration mouse various dose takes mouse after one month
Thymus gland and spleen are weighed, and calculate dirty body ratio, and dirty body is met homogeneity of variance requirement, use single factor test than carrying out homogeneity test of variance
The comparative approach two-by-two of mean carries out statistical disposition between multiple test groups and a control group in variance analysis method.By 7 knot of table
Fruit as it can be seen that 0.5ml/kg/d, 1.0ml/kg/d, 3.0ml/kg/d group thymus gland body ratio and spleen body ratio compared with 0ml/kg/d groups,
There is increase.
The influence of table 7 Chinese medicine composition Oral Liquid On Mice thymus gland, spleen organ of the present invention
The influence for the mouse spleen lymphocyte conversion that 2.3 Chinese medicine composition oral liquids of the present invention induce ConA
The Chinese medicine composition oral liquid of the present invention of orally administration mouse various dose carries out ConA after one month with mtt assay
The mouse spleen lymphocyte transformation experiment of induction, calculates plus ConA holes are with being not added with the difference of ConA holes absorbance, and it is carried out
Homogeneity test of variance, meets homogeneity of variance requirement, between multiple test groups in one-way analysis of variance method and a control group
The comparative approach two-by-two of mean carries out statistical disposition.By 8 result of table as it can be seen that 1.0ml/kg/d, 3.0ml/kg/d group ConA holes with
It is not added with ConA holes absorbance difference higher than 0ml/kg/d groups to compare, difference is statistically significant (P ＜ 0.05).
The influence of the Chinese medicine composition Oral Liquid On Mice Splenic vein hemodynamics of the present invention of table 8
Influence of the 2.4 Chinese medicine composition oral liquids of the present invention to DNFB inducing mouses DTH
The Chinese medicine composition oral liquid of the present invention of orally administration mouse various dose is carried out after one month with ear swelling method
DNFB inducing mouses DTH is tested, and calculates auricular concha weightening, and carries out homogeneity test of variance to it, meets homogeneity of variance requirement, with list
The comparative approach two-by-two of mean carries out statistical disposition between multiple test groups and a control group in analysis of variance method.By table
For 9 results as it can be seen that the weightening of 3.0ml/kg/d groups auricular concha is compared higher than 0ml/kg/d groups, difference is statistically significant (P ＜ 0.05).
Influence of the Chinese medicine composition oral liquid of the present invention of table 9 to DNFB inducing mouses DTH
The influence of 2.5 Chinese medicine composition Oral Liquid On Mice antibody-producting cells (hemolysis plaque number) of the present invention
The Chinese medicine composition oral liquid of the present invention of orally administration mouse various dose improves slide after one month, with Jerne
Method carries out mouse antibodies cellulation experiment, calculates hemolysis plaque number, and carries out homogeneity test of variance to it, meets homogeneity of variance
It is required that counted with the comparative approach two-by-two of mean between multiple test groups in one-way analysis of variance method and a control group
Processing.By 10 result of table as it can be seen that 3.0ml/kg/d groups hemolysis plaque number compares higher than 0ml/kg/d groups, difference is statistically significant
(P ＜ 0.05).
The influence of the Chinese medicine composition Oral Liquid On Mice hemolysis plaque number of the present invention of table 10
2.6 Chinese medicine composition Oral Liquid On Mice half hemolytic dose (HC of the present invention50) influence
The Chinese medicine composition oral liquid of the present invention of orally administration mouse various dose is surveyed after one month with half hemolytic value method
Determine the half hemolytic dose (HC of mouse50), and homogeneity test of variance is carried out to it, meet homogeneity of variance requirement, use single factor test
The comparative approach two-by-two of mean carries out statistical disposition between multiple test groups and a control group in variance analysis method.By 11 knot of table
Fruit is as it can be seen that 3.0ml/kg/d group mice serum half hemolytic values (HC50) compare higher than 0ml/kg/d groups, difference is statistically significant
(P ＜ 0.05).
The Chinese medicine composition Oral Liquid On Mice HC of the present invention of table 1150Influence
The influence of 2.7 Chinese medicine composition Oral Liquid On Mice carbonic clearance abilities of the present invention
It is real to carry out mouse carbonic clearance after one month for the Chinese medicine composition oral liquid of the present invention of orally administration mouse various dose
Test, calculate phagocytic index a, and homogeneity test of variance is carried out to it, meet homogeneity of variance requirement, with one-way analysis of variance method
In between multiple test groups and a control group mean comparative approach two-by-two carry out statistical disposition.By 12 result of table as it can be seen that
0.5ml/kg/d, 1.0ml/kg/d, 3.0ml/kg/d group mouse phagocytic index a and 0ml/kg/d group, which more have, to be increased.
The influence of the Chinese medicine composition Oral Liquid On Mice carbonic clearance ability of the present invention of table 12
The phagocytic percentage of 2.8 Chinese medicine composition Oral Liquid On Mice peritoneal macrophage phagocytosis chicken red blood cells of the present invention
And the influence of phagocytic index
The Chinese medicine composition oral liquid of the present invention of orally administration mouse various dose is carried out small after one month with half intracorporal method
The phagocytosis chicken red blood cell experiment of mouse peritoneal macrophage, calculates phagocytic index and phagocytic percentage, and by phagocytic percentage through sin- 1P1/2Homogeneity test of variance is carried out after (P is phagocytic percentage, is decimally represented) conversion, phagocytic index and phagocytic percentage meet
Homogeneity of variance requirement, with the comparative approach two-by-two of mean between multiple test groups in one-way analysis of variance method and a control group
Carry out statistical disposition.By 13 result of table as it can be seen that 0.5ml/kg/d, 1.0ml/kg/d, 3.0ml/kg/d group mouse peritoneal macrophage are thin
Endocytosis bite chicken red blood cell phagocytic percentage and phagocytic index compared with 0ml/kg/d groups, have increase.
The Chinese medicine composition Oral Liquid On Mice peritoneal macrophage phagocytosis chicken red blood cell phagocytic rate of the present invention of table 13 and phagocytosis
The influence of index
The influence of 2.9 Chinese medicine composition Oral Liquid On Mice NK cytoactives of the present invention
The Chinese medicine composition oral liquid of the present invention of orally administration mouse various dose is after one month, with lactic dehydrogenase enzyme process into
Row NK cells in mice determination of activity, by NK cytoactives through sin-1P1/2(P is NK cytoactives, is decimally represented) conversion is laggard
Row homogeneity test of variance, meets homogeneity of variance requirement, with multiple test groups in one-way analysis of variance method and a control group
Between mean comparative approach two-by-two carry out statistical disposition.By 14 result of table as it can be seen that 3.0ml/kg/d group NK cytoactives are higher than
0ml/kg/d groups compare, and difference is statistically significant (P ＜ 0.05).
The influence of the Chinese medicine composition Oral Liquid On Mice NK cytoactives of the present invention of table 14
According to《Health food is examined and assessment technique specification》Strengthen immunity function test result judgement, the present invention
Chinese medicine composition has the function of strengthen immunity.
Effect example 3
Chinese medicine composition safety testing of the present invention
1 materials and methods
1.1 sample：3 gained capsule of the embodiment of the present invention, specification：0.5g/, lot number：20131006.Sample morphology
For brown color liquid, put and preserved at shady and cool dry and ventilated.
1.2 experimental animal：Cleaning grade healthy adult ICR mouse, half male and half female, the weight of animals female is 18.5-20.9g,
There is provided by Yangzhou University's comparative medicine center, production licence number SCXK (Soviet Union) 2012-0004, quality certification number：0025266.Clearly
Clean level health weanling rat, half male and half female, is provided by Shanghai Slac Experimental Animal Co., Ltd., production licence number
SCXK (Soviet Union) 2012-0002, quality certification number：2007000570051.Experimental animal feeding temperature is 20-25 DEG C, and relative humidity is
40-70%
1.3 key instrument：T-1000 type electronic balances, JJ-100 types electronic balance, JA-2003 electronic balances, AL104
Type electronic balance, centrifuge, OLYMPUS microscopes, 7100 type automatic clinical chemistry analyzer of Hitachi, BAKERSG-603 biology peaces
Full cabinet, electro-heating standing-temperature cultivator, SX-700 autoclaves, KYC-100C types constant-temperature table, SIEMENS ADVIA2120i blood
Liquid analyzer.Salmonella typhimurium saltant TA97, TA98, TA100, TA102 derive from Fudan University's public health
Institute；S-9 derives from Fudan University's School of Public Health.
1.4 experimental method
1.4.1 acute toxicity test in mice：Acute toxicity test in mice is carried out with maximal tolerance dose method
1.4.2 30 days feeding trials of rat：After each group animal gives the continuous nursing of basal feed 30 days, fasting overnight, by
Only weigh, 10% chloraldurate presses 3.5ml/kg intraperitoneal injection of anesthesia, its hematological indices and blood biochemical are taken through abdominal aorta
Index, observes 1. general behavior performance and death condition；2. weight, food utilization；3. hematological indices；4. blood biochemical
Index；5. organ coefficient；6. pathological examination.
1.4.3 Salmonella reversion test：Press《Health food is examined and assessment technique specification》Version in 2003, is mixed using dressing plate
Enter method, tested after selecting the identified qualification of salmonella typhimurium saltant TA97, TA98, TA100, TA102.
1.4.4 mouse marrow cell micro nuclear test：25-30gICR cleaning grades mouse 50 is selected, adapts to environment after 3 days
Tested.5 groups are randomly divided into by weight, every group 10, half male and half female.If the 10th, 5,2.5ml/kg3 dosage group, feminine gender
Control group (sterile water, 20ml/kg gavages), positive controls (endoxan, 40mg/kg intraperitoneal injections).Given the test agent nothing
Bacterium water is prepared, and concentration containing given the test agent is 50%, 25%, 12.5% to high, medium and low dosage respectively, with 20ml/kg gavages, is used
30h gives tested material method.Give given the test agent interval 24h twice, second to after given the test agent 6 it is small when put to death animal, take breastbone normal
Regulation piece, microscopy, every mouse count 1000 Polychromatic erythrocytes (PCE), polychromatic erythrocyte number of the observation containing micronucleus simultaneously
Microkernel incidence is calculated, in terms of permillage, mature erythrocyte number observed during 200 polychromatic erythrocytes is counted per mouse,
And calculate polychromatic erythrocyte and mature erythrocyte ratio (PCE/RBC).
1.4.5 mouse inbred strain：Weight 25-30gICR cleaning grades mouse 25 is selected, adapts to environment after 3 days
Tested.5 groups are randomly divided into by weight, every group 5.If the 10th, 5,2.5ml/kg3 dosage group, negative control group are (sterile
Water, 20ml/kg gavages), positive controls (endoxan, 40mg/kg intraperitoneal injection).Given the test agent is prepared with sterile water, high,
In, low dosage respectively concentration containing given the test agent be 50%, 25%, 12.5%, with 20ml/kg gavages, 1 time a day, continuous 5 days.
Put to death animal within the 35th day after given the test agent is given first, take both sides epididymal sperm filtrate routinely film-making, microscopy.Counted per mouse
The sperm of 1000 structural integrities, calculates Sperm malformation rate (with percentage).
1.5 data processing：Chmice acute Oral toxicity test data carries out data statistics point using SPSS10.0 softwares
Analysis；30 days feeding trials of rat carry out homogeneity test of variance using SPSS16.0 softwares to each experiment initial data；Micronucleus test
As a result examined using Poisson distributions U and carry out statistical disposition；Mouse inbred strain result is counted using rank sum test
Processing.
2 results
2.1 acute toxicity test in mice
During experimental observation, each group animal diet followed and activity are normal, well-grown, have no any poisoning manifestations, no death.
Influence of the Chinese medicinal composition capsules of the present invention to mouse weight and death toll is shown in Table 15.The result shows that Chinese medicine composition glue of the present invention
Capsule is female, male its mouse oral MTD is more than 22400mg/kg, and according to acute toxicity dose grading standard, which belongs to nontoxic level.
The influence of the Chinese medicine composition Oral Liquid On Mice acute oral toxicity of the present invention of table 15
2.2 feeding trials of rat 30 days
2.2.1 general behavior performance and death condition：Chinese medicine composition of the present invention is with 3.0ml/kg/d, 6.5ml/kg/
D, the dosage of 10.0ml/kg/d (30 times, 65 times, 100 times equivalent to human body recommended dose) gives rat 30 days, each group animal
General appearance and behavior are showed no obvious abnormalities, without poisoning manifestations, without death.
2.2.2 Chinese medicine composition of the present invention influences rat body weight, food utilization：Chinese medicine composition of the present invention with
The dosage of 3.0mg/kg/d, 6.5mg/kg/d, 10.0mg/kg/d (30 times, 65 times, 100 times equivalent to human body recommended dose)
Give rat 30 days, each dosage group rat the 30th day weight, total foodstuff utilization rate are compared with 0mg/kg/d groups, and there was no significant difference
(P ＞ 0.05).It the results are shown in Table 16- tables 19.
Influence of the Chinese medicine composition oral liquid of the present invention of table 16 to rat body weight
The influence that the Chinese medicine composition oral liquid of the present invention of table 17 increases weight rat body weight
The food ration of the Chinese medicine composition oral liquid each group rat of the present invention of table 18
Influence of the Chinese medicine composition oral liquid of the present invention of table 19 to rat chow utilization rate
2.2.3 influence of the Chinese medicine composition of the present invention to rat blood index：Orally administration rat Chinese medicine group of the present invention
Compound oral liquid 30 days, measures each dosage group rat blood red blood cell count(RBC), hemoglobin level, white blood cell count(WBC) and its classification
Etc. index.By table 20-1 to table 20-2 as it can be seen that each dosage group of indices is compared with 0mg/kg/d groups, difference is anticipated without statistics
Adopted (P ＞ 0.05).
Influence of the table 20-1 Chinese medicine composition oral liquids of the present invention to rat blood index
Influence of the table 20-2 Chinese medicine composition oral liquids of the present invention to rat blood index
2.2.4 influence of the Chinese medicine composition of the present invention to rat blood biochemical indicator：Orally administration rat various dose
Chinese medicine composition oral liquid of the present invention 30 days, measure each dosage group rat blood serum glutamic-pyruvic transaminase, glutamic-oxalacetic transaminease, total protein,
Albumin, T-CHOL, triglycerides, urea nitrogen, the level of creatinine and blood glucose.By table 21 as it can be seen that female rats 3.0mg/kg/
D groups creatinine and male rat 6.5,10.0mg/kg/d group total proteins are above 0mg/kg/d groups (P ＜ 0.05), though above numerical value
There is a significant difference, but numerical value is in this laboratory history normal range (NR), and without dose-response relationship, be not considered as to have bad
Biological significance.Other each dosage group items blood parameters are compared with 0mg/kg/d groups, the not statistically significant (P of difference
＞ 0.05).
Influence of the table 21-1 Chinese medicine composition oral liquids of the present invention to blood biochemistry of rats
Note：* compared with 0ml/kg/d, P ＜ 0.05
Influence of the table 21-2 Chinese medicine composition oral liquids of the present invention to blood biochemistry of rats
Note：* compared with 0mg/kg/d, P ＜ 0.05
2.2.5 influence of the Chinese medicine composition of the present invention to Rats Organs and Tissues absolute weight and relative weight：Orally administration rat
The Chinese medicine composition oral liquid of the present invention of various dose 30 days, measures each dosage group rat liver, kidney, spleen, testis, ovary
Absolute weight and relative weight (dirty/body).By table 22 as it can be seen that the indices and 0mg/kg/d groups of each dosage group of mouse female, great and mighty or powerful
Compare, difference is not statistically significant (P ＞ 0.05).
Table 22-1 Chinese medicine composition oral liquid each group Rats Organs and Tissues absolute weights of the present invention and relative weight
Table 22-2 Chinese medicine composition oral liquid each group Rats Organs and Tissues absolute weights of the present invention and relative weight
2.2.6 pathological examination
Each group rat gross anatomy is showed no abnormal change.Microscopy result is as follows：
0mg/kg/d groups female rats totally 10：
1. liver：Lobuli hepatis structure is clear, liver rope marshalling；Liver cell size is basically identical, and nucleus size is homogeneous,
Nuclear membrane, kernel understand, cytoplasm even dyeing, sinus hepaticus is slight hemostasis 1.
2. kidney：Kidney skin, medullary substance boundary clear, nephron structure is normal, and glomerulus is without inflammatory change, fibrosis, glass
Sample lesion etc. changes, and renal tubule also has no obvious lesion,
3. spleen：Red, white pulp understands that central artery occupies acini lienalis center, and corpusculum is without obvious atrophy, snius lienis expansion, spleen
Girder is normal.
4. Stomach duodenum：Show that mucous membrane is normal, no inflammatory changes and rotten to the corn, ulcer is formed.
5. ovary：The corpus luteum or lean type formed in ovary after the ovarian follicle of visible different developmental phases and ovulation.
0mg/kg/d groups male rat totally 10：
1. liver：Lobuli hepatis structure is clear, liver rope marshalling；Liver cell size is basically identical, and nucleus size is homogeneous,
Nuclear membrane, kernel understand, cytoplasm even dyeing, slight hepatic cell steatosis 3.
2. kidney：Kidney skin, medullary substance boundary clear, nephron structure is normal, and glomerulus is without inflammatory change, fibrosis, glass
Sample lesion etc. changes, and renal tubule also has no obvious lesion,
3. spleen：Red, white pulp understands that central artery occupies acini lienalis center, and corpusculum is without obvious atrophy, snius lienis expansion, spleen
Girder is normal.
4. Stomach duodenum：Show that mucous membrane is normal, no inflammatory changes and rotten to the corn, ulcer is formed.Under mucous membrane, muscle layer, serous coat
Layer changes without inflammatory.
5. testis：Convoluted tubule of testis is made of sertoli cell and epithelial cell, primary, secondary spermatocyte, spermatid
Development is normal.
10mg/kg/d groups female rats totally 10：
1. liver：Lobuli hepatis structure is clear, liver rope marshalling；Liver cell size is basically identical, and nucleus size is homogeneous,
Nuclear membrane, kernel understand, cytoplasm even dyeing, slight hepatic cell steatosis 1.
2. kidney：Kidney skin, medullary substance boundary line understand that nephron structure is normal, and glomerulus is without inflammatory change, fibrosis, glass
Sample lesion etc. changes, and renal tubule also has no obvious lesion,
3. spleen：Red, white pulp understands that central artery occupies acini lienalis center, and corpusculum is without obvious atrophy, snius lienis expansion, spleen
Girder is normal.
4. Stomach duodenum：Show that mucous membrane is normal, no inflammatory changes and rotten to the corn, ulcer is formed.Under mucous membrane, muscle layer, serous coat
Layer changes without inflammatory.
5. ovary：The corpus luteum or lean type formed in ovary after the ovarian follicle of visible different developmental phases and ovulation.
10mg/kg/d groups male rat totally 10：
1. liver：Lobuli hepatis structure is clear, liver rope marshalling；Liver cell size is basically identical, and nucleus size is homogeneous,
Nuclear membrane, kernel understand, cytoplasm even dyeing, slight hepatic cell steatosis 2.
2. kidney：Kidney skin, medullary substance boundary line understand that nephron structure is normal, and glomerulus is without inflammatory change, fibrosis, glass
Sample lesion etc. changes, and renal tubule also has no obvious lesion,
3. spleen：Red, white pulp understands that central artery occupies acini lienalis center, and corpusculum is without obvious atrophy, snius lienis expansion, spleen
Girder is normal.
4. Stomach duodenum：Show that mucous membrane is normal, no inflammatory changes and rotten to the corn, ulcer is formed.Under mucous membrane, muscle layer, serous coat
Layer changes without inflammatory.
5. testis：Convoluted tubule of testis is made of sertoli cell and epithelial cell, primary, secondary spermatocyte, spermatid
Development is normal.
To sum up, liver,kidney,spleen caused by Chinese medicine composition of the present invention, Stomach duodenum, testis, ovary internal organs toxicity are had no
Pathological change.
2.3 Salmonella reversion test：By table 23, table 24 as it can be seen that Chinese medicine composition of the present invention each dosage group time change clump count does not surpass
2 times that negative control group returns change clump count are crossed, also without dose-response relationship.And Chinese medicine composition of the present invention is to mouse typhus sramana
Tetra- plants of test strains of Salmonella TA97, TA98, TA100, TA102, when adding and being not added with S-9, are showed no and cause gene mutation effect.
The Chinese medicine composition oral liquid Salmonella reversion test of the present invention of table 23, which returns, becomes clump count(first time)
The Chinese medicine composition oral liquid Salmonella reversion test of the present invention of table 24, which returns, becomes clump count(for the second time)
2.4 mouse marrow cell micro nuclear test：By table 25 as it can be seen that each dosage group of Chinese medicine composition oral liquid of the present invention
There was no significant difference compared with negative control group for micronuclear rates (P ＞ 0.05), and positive controls are then significantly higher than negative control group
(P ＜ 0.01).Have no Chinese medicine composition Oral Liquid On Mice Micronucleus formation of the present invention and PCE/RBC ratios
Value has an impact.
The Chinese medicine composition Oral Liquid On Mice bone marrow cell micronucleus incidence of the present invention of table 25 and polychromatic erythrocyte with into
The influence of ripe red blood cell ratio (PCE/RBC)
Note：Compared with negative control group, P ＜ 0.01
2.5 mouse inbred strain：By table 26, table 27 as it can be seen that each dosage group rate of teratosperm and negative control group ratio
Compared with there was no significant difference (P ＞ 0.05), and positive controls mouse sperm deformity rate relatively has the conspicuousness poor with negative control group
Different (P ＜ 0.01).It can be seen that Chinese medicine composition Oral Liquid On Mice rate of teratosperm of the present invention does not have an impact.
The influence of the Chinese medicine composition Oral Liquid On Mice rate of teratosperm of the present invention of table 26
Note：Compared with negative control group, P ＜ 0.01
The influence of the Chinese medicine composition Oral Liquid On Mice teratospermia type of the present invention of table 27 and ratio
Effect example 4
Chinese medicine composition of the present invention improves sleep human feeding trial
1 test method：Using itself between group two kinds of control designs, select 120 simple long-term with insomnia
Patient, at 18~60 years old age, test-meal group and control group are randomly divided into according to symptom weight, every group 60, test-meal group male 26,
Women 34, average age are 52.75 ± 10.25 years old, control group male 29, women 31, average age for 51.26 ±
9.79.Test-meal group：The Chinese medicinal composition granules that the embodiment of the present invention 2 is prepared are taken, control group eats placebo, comfort
Agent appearance, color and luster, weight, instructions of taking are identical with the present invention.Everyone takes 3 times subject daily, 1 bag every time (5g/ bags), with temperature
Taken with boiled water.Normal diet during experiment, does not change original eating habit, and disables the medicine and Chinese medicine of other treatment insomnia
Composition, is 90 days during observation.
Symptom integral is observed：By the weight situation scores accumulated of insomnia (severe 3 is divided, moderate 2 is divided, 1 point slight).Two
There was no significant difference before the treatment (P ＞ 0.05) for group symptom integral.Two groups of blood, urine, feces routine and hepatic and renal function, chest before test-meal
Thoroughly, the inspection such as electrocardiogram, B ultrasound is in normal range (NR).
2 results
Itself compare before and after test-meal group test-meal and (P ＜ 0.01) be obviously reduced in control group comparison among groups, clinical symptoms integration,
Test-meal group crowd's sleep quality is obviously improved, and does not report other side effects, and Chinese medicine composition of the present invention has the work(for improving and sleeping
Energy.It the results are shown in Table 28.
Clinical symptoms integrate before and after 28 test-meal of table
* is compared with before test-meal, P ＜ 0.01；##Comparison among groups, P ＜ 0.01.
Effect example 5
Chinese medicine composition strengthen immunity human feeding trial of the present invention
1 test method：120 simple immunocompromised patients are selected, examination is randomly divided into by subject immune's power situation
Food group and control group, consider to influence principal element such as age, gender, diet etc. of result as far as possible, carry out balanced by every group 60
Property examine, with the comparativity between guarantee group.Test-meal group male 32, women 28, average age 48.72 ± 10.01 years old；Control
Group male 27, women 33, average age 47.45 ± 9.87 years old.Test-meal group：Take 4 gained Chinese medicine group of the embodiment of the present invention
Compound tablet, control group eat placebo, and placebo appearance, color and luster, weight, instructions of taking are identical with the present invention.Subject is every
People takes 3 times daily, and 2 tablets once, uses warm water delivery service.Normal diet during experiment, does not change original eating habit, continuous clothes
With 90 days.
The inspection such as two groups of blood before test-meal, urine, feces routine and hepatic and renal function, Chest X-rays, electrocardiogram, B ultrasound is in normal range (NR), and two
The equal no significant difference of content (P ＞ 0.05) of group subject's lymphocyte and macrophage, is comparable.
2 results
90 days after taking, the content of lymphocyte and macrophage substantially increases (P ＜ 0.01) before and after test-meal group test-meal, bright
Aobvious to be higher than control group, two comparison among groupss have significant difference (P ＜ 0.01).The result shows that examination takes Chinese medicine composition 90 of the present invention
After it, test-meal group test-meal is front and rear itself compare and test-meal after test-meal group and control group comparison among groups, lymphocyte and macrophage
Content substantially increase (P ＜ 0.01), illustrate the invention have enhancing immunity of organisms function.It the results are shown in Table 29- tables 30.
Blood lymphocytes change before and after 29 test-meal of table
Blood macrophage changes before and after 30 test-meal of table
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (13)
1. a kind of have improvement sleep, the Chinese medicine composition of strengthen immunity effect, it is characterised in that it is by following parts by weight
Raw material be made：
2~10 parts of wilsonii, 1~10 part of American Ginseng, 5~30 parts of spina date seed, 5~20 parts of sealwort, 1~10 part of psoralea corylifolia, Poria cocos 5
~30 parts.
2. according to claim 1 have improvement sleep, the Chinese medicine composition of strengthen immunity effect, it is characterised in that
It is made of the raw material of following parts by weight：
4~8 parts of wilsonii, 2~8 parts of American Ginseng, 10~20 parts of spina date seed, 6~16 parts of sealwort, 2~8 parts of psoralea corylifolia, Poria cocos 10
~20 parts.
3. according to claim 1 have improvement sleep, the Chinese medicine composition of strengthen immunity effect, it is characterised in that
It is made of the raw material of following parts by weight：
2 parts of wilsonii, 1 part of American Ginseng, 10 parts of spina date seed, 6 parts of sealwort, 3 parts of psoralea corylifolia, 10 parts of Poria cocos.
4. according to claim 1 have improvement sleep, the Chinese medicine composition of strengthen immunity effect, it is characterised in that
It is made of the raw material of following parts by weight：
4 parts of wilsonii, 2 parts of American Ginseng, 30 parts of spina date seed, 10 parts of sealwort, 8 parts of psoralea corylifolia, 30 parts of Poria cocos.
5. according to claim 1 have improvement sleep, the Chinese medicine composition of strengthen immunity effect, it is characterised in that
It is made of the raw material of following parts by weight：
4 parts of wilsonii, 8 parts of American Ginseng, 20 parts of spina date seed, 16 parts of sealwort, 2 parts of psoralea corylifolia, 10 parts of Poria cocos.
6. according to claim 1 have improvement sleep, the Chinese medicine composition of strengthen immunity effect, it is characterised in that
It is made of the raw material of following parts by weight：
6 parts of wilsonii, 6 parts of American Ginseng, 5 parts of spina date seed, 5 parts of sealwort, 7 parts of psoralea corylifolia, 5 parts of Poria cocos.
7. according to claim 1 have improvement sleep, the Chinese medicine composition of strengthen immunity effect, it is characterised in that
It is made of the raw material of following parts by weight：
8 parts of wilsonii, 2 parts of American Ginseng, 15 parts of spina date seed, 7 parts of sealwort, 3 parts of psoralea corylifolia, 20 parts of Poria cocos.
8. according to claim 1 have improvement sleep, the Chinese medicine composition of strengthen immunity effect, it is characterised in that
It is made of the raw material of following parts by weight：
6 parts of wilsonii, 5 parts of American Ginseng, 15 parts of spina date seed, 11 parts of sealwort, 5 parts of psoralea corylifolia, 15 parts of Poria cocos.
9. according to claim 1 have improvement sleep, the Chinese medicine composition of strengthen immunity effect, it is characterised in that
It is made of the raw material of following parts by weight：
5 parts of wilsonii, 5 parts of American Ginseng, 11 parts of spina date seed, 14 parts of sealwort, 3 parts of psoralea corylifolia, 18 parts of Poria cocos.
10. a kind of have improvement sleep, the pharmaceutical preparation of strengthen immunity effect, it is characterised in that by claim 1~9 times
The extract and pharmaceutically acceptable carrier of Chinese medicine composition described in one are prepared into oral liquid, granule, capsule, piece
Agent or pill.
11. claim 1~9 any one of them, which has, improves sleep, the preparation for the Chinese medicine composition that strengthen immunity acts on
Method, it is characterised in that comprise the following steps：
A, by claim 1~9 any one of them parts by weight, American Ginseng is taken, wilsonii, psoralea corylifolia, adds medicinal material weight 1
~10 times amount volume 10~90% ethanol, heating and refluxing extraction 1~3 time, every time 1~3 it is small when, merge to obtain alcohol extracting
Liquid；
B, take step A ethanol extract after the spina date seeds of the dregs of a decoction and claim 1~9 any one of them parts by weight, sealwort,
Poria cocos, adds the water measured again of 1~15 medicinal material, heating extraction 1~3 time, every time 1~3 it is small when, merge to obtain aqueous extract；
C, step A alcohol extracts are concentrated into medicinal material equivalent as 0.8:1~2:1, step B Aqueous extracts are concentrated into medicinal material equivalent and are about
0.8:1~2:1, mixing, it is about 1 to continue to be concentrated into medicinal material equivalent:1~2:1
D, the one or more in concentrate obtained by step C and chitosan, xylitol, Sucralose, dextrin and starch are combined,
Oral liquid, granule, capsule, tablet or pill is made.
12. the preparation method of the Chinese medicine composition according to claim 11 that there is improvement sleep, strengthen immunity to act on,
It is characterised in that it includes following steps：
Claim 1~9 any one of them parts by weight are pressed in step A, take American Ginseng, wilsonii, psoralea corylifolia, adds medicinal material
50% ethanol of 5 times of volumes of weight, heating and refluxing extraction 2 times, every time 2 it is small when, merge to obtain alcohol extract；
Taken in step B the spina date seeds of any one of step A ethanol the extraction dregs of a decoction and claim 1~9 parts by weight, sealwort and
Poria cocos, adds the water of total 10 times of volumes of medicinal material, and heating decocts extraction 2 times, every time 2 it is small when, merge to obtain aqueous extract；
In step C, step A alcohol extracts are concentrated into medicinal material equivalent as 1:1, it is about 1 that step B Aqueous extracts, which are concentrated into medicinal material equivalent,:
1, after mixing, it is about 1.5 to continue to be concentrated into medicinal material equivalent:1.
13. having described in claim 1~9 any one improves sleep, the Chinese medicine composition of strengthen immunity effect is being made
The standby application having in the medicine or/and health products for improving sleep, strengthen immunity effect.
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| CN106177389B (en) * | 2016-08-22 | 2020-10-30 | 宁波御坊堂生物科技有限公司 | Traditional Chinese medicine preparation with functions of relaxing bowel, improving sleep and enhancing immunity and preparation method thereof |
| CN106360716A (en) * | 2016-08-22 | 2017-02-01 | 蓬莱海洋（山东）股份有限公司 | Traditional Chinese medicinal composition for improving body immunity and sleep, and preparation method thereof |
| CN114788845A (en) * | 2021-01-26 | 2022-07-26 | 四川福生源科技有限公司 | Traditional Chinese medicine composition with sleep improving effect and preparation method and application thereof |
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| CN102697047A (en) * | 2012-06-11 | 2012-10-03 | 宁夏红中宁枸杞制品有限公司 | Method for preparing oral liquid of Chinese wolfberry |
| CN102948751A (en) * | 2012-11-23 | 2013-03-06 | 乔德京 | Ginseng tuckahoe acanthopanax senticosus capsule |
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| CN102948751A (en) * | 2012-11-23 | 2013-03-06 | 乔德京 | Ginseng tuckahoe acanthopanax senticosus capsule |
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Denomination of invention: Traditional Chinese medicine compositions with the effects of improving sleep and enhancing immunity and their preparation methods and applications Effective date of registration: 20221019 Granted publication date: 20180508 Pledgee: Shanghai Bank Co.,Ltd. Suzhou Branch Pledgor: LEIYUNSHANG PHARMACEUTICAL GROUP Co.,Ltd. Registration number: Y2022320010593 |