CN104998083A - Traditional Chinese medicine composition capable of improving immunity, and preparation method and applications thereof - Google Patents

Abstract

The invention discloses a traditional Chinese medicine composition capable of improving immunity, and a preparation method and applications thereof. The traditional Chinese medicine composition is prepared by taking 150 to 200g of American ginseng, 30 to 50g of hairy antler, 300 to 400g of rhodiola rosea, 300 to 400g of the fruit of Chinese wolfberry, and 100 to 150g of dendrobium officinale as main raw materials. According to the preparation method, dendrobium officinale, the fruit of Chinese wolfberry, and rhodiola rosea are decocted with 6 to 10 times of water for 1 to 1h, and decoction is carried out for 1 to 3 times; an obtained solution is filtered, and obtained filtrate is collected and is subjected to concentration at 60 DEG C so as to obtain an extract with a relative density ranging from 1.05 to 1.15; the extract is subjected to spray drying and sieving so as to obtain a dried paste powder; the dried paste powder is mixed with American ginseng powder and hairy antler powder so as to obtain a mixed powder; and an oral preparation is prepared via a conventional method. It is shown by animal experiment results that the traditional Chinese medicine composition is capable of relieving physical fatigue, and improving immunity, is rapid to be absorbed, and is convenient for consumption and carrying.

Description

Chinese medicine composition of a kind of enhancing immunity and preparation method thereof and application

Technical field

The invention belongs to technical field of traditional Chinese medicine preparation, relate to the Chinese medicine preparation of enhancing immunity, alleviating physical fatigue, is Chinese medicine composition of a kind of enhancing immunity and preparation method thereof and application in particular.

Background technology

Fatigue is a kind of physiological phenomenon, is a kind of protective mechanism concerning people.Fatigue is that health sends the signal had a rest to us.If no matter ignored it, health will suffer damage, and finally falls sick from overwork.The tired crowd of easy generation: the urbanite of age between 20 ~ 45 years old, this age bracket because of work, study, family's pressure is increasing and cause body and mind anxiety tired out; Mature women, life is nervous, and family burden is heavier, and health is weak compared with male, easily causes tired out, Somatic discomfort and spirit and psychological pressure overweight; Long-term work is nervous, is in the crowd under the large working environment of competitive pressure; Suddenly the people that momentous life events (as divorce, disease) affects is suffered; Live irregular, diet and the irrational crowd of trophic structure; The condition subject such as anxiety neurosis, depression.Fatigue is modal health problem in Modern human populations, and the people of each ages has and to be a great deal ofly stranded by this.Along with the quickening of modern life rhythm, social competition is fierce, and survival pressure is large, and the puzzlement of this symptom is more and more outstanding.The suitable population of alleviating physical fatigue comprises: athlete and be keen on sports, the crowd of body-building; Work in hot environment personnel; Military activity personnel; Highlands operating personnel and all kinds of crowds that easily generation is tired.

In recent years, along with improving constantly of living standard, the health of people to self is more and more paid attention to, and suitably takes some health foods and keeps fit and become the important means of the countries in the world common people gradually.Modern life rhythm is accelerated, and social competition aggravates, and a lot of people, because the pressure etc. of work, study, to give rise to diseases because of hypoimmunity and fatiguability problem becomes the health problem perplexing a lot of people.Therefore develop the health food with enhancing immunity and alleviating physical fatigue to be with a wide range of applications.At present, on market, the health product of enhancing immunity function mainly contain following a few class:

The first kind is ginseng class health product, is usually reached maintain body normal physiological function, the object of relieving fatigue by the nutrition consumed in supplementary motion.Equations of The Second Kind is vitamins health product, supplements vitamin and the trace element of needed by human.3rd class is microelement kind health product, and it is the function of function, particularly blood circulation by improving biological organs, and the removing of acceleration bodies intracellular metabolite material, discharge, reach resisting fatigue object.A lot of Chinese herbal and crude drugs preparations all belongs to this class.4th class is the product of the raising immunity function that some other is declared.The health food of enhancing immunity function in health food market in occupation of sizable share.Through consulting State Food and Drug Administration health food official written reply data base, have enhancing immunity, alleviating physical fatigue product more, but the national OTC simultaneously with the two function has about 200, import health food is less than 10, dosage form mostly is tablet, oral liquid, capsule, also has the dosage form such as electuary, tea beverage.Therefore develop the health food with enhancing immunity and alleviating physical fatigue to be with a wide range of applications.

Summary of the invention

The present invention for primary raw material, adopts suitable technique to make various dosage form with Radix Panacis Quinquefolii, cervus elaphus linnaeus, Radix Rhodiolae, Fructus Lycii, Herba Dendrobii, has to absorb fast, the feature such as to take, easy to carry.Raw materials used ample supply and prompt delivery, to be easy to get; Technique is simply controlled, and quality standard is higher, can ensure the quality of product.Also meet the requirement of State Food and Drug Administration to health food, safe toxicity test has no toxic and side effects, has higher edible safety, can long-term taking simultaneously.Product orientation is accurate, with the obvious advantage, has considerable development prospect.

For achieving the above object, the invention discloses following technology contents:

A Chinese medicine composition for enhancing immunity, is characterized in that it is made up of the raw material of Chinese medicine of following weight:

Radix Panacis Quinquefolii 150-200g

Cervus elaphus linnaeus 30-50g

Radix Rhodiolae 300-400g

Fructus Lycii 300-400g

Herba Dendrobii 100-150g.

The Chinese medicine composition of preferred enhancing immunity, is characterized in that it is made up of the raw material of Chinese medicine of following weight:

Radix Panacis Quinquefolii 185-190g

Cervus elaphus linnaeus 35-40g

Radix Rhodiolae 370-380g

Fructus Lycii 370-380g

Herba Dendrobii 120-130g.

The most preferably Chinese medicine composition of enhancing immunity, is characterized in that it is made up of the raw material of Chinese medicine of following weight:

Radix Panacis Quinquefolii 187.5g

Cervus elaphus linnaeus 37.5g

Radix Rhodiolae 375g

Fructus Lycii 375g

Herba Dendrobii 125g.

Chinese medicine composition of the present invention, refers to oral formulations, such as pill, granule, tablet, soft extract, syrup, mixture, drop pill, capsule, medicated wine, tincture.

The present invention further discloses the preparation method of the Chinese medicine composition of enhancing immunity, it is characterized in that being undertaken by following step:

(1) get the Herba Dendrobii of formula proportion, Fructus Lycii, Radix Rhodiolae add 6 ~ 10 times amount soak by water 1 ~ 3 time, each 1 ~ 2 hour, filter, merging filtrate, filtrate concentrates, and is concentrated into the extractum of 60 DEG C of relative densities 1.05 ~ 1.15;

(2) get extractum and carry out spraying dry, sieve, get dry extract powder;

(3) get dried cream powder, Radix Panacis Quinquefolii powder, the mixing of cervus elaphus linnaeus powder, obtain mixed powder; Technology makes oral formulations routinely.

The present invention further discloses the application of enhancing immunity Chinese medicine composition in preparation treatment enhancing immunity, alleviating physical fatigue medicine.Experimental result shows: compositions has the function of alleviating physical fatigue, enhancing immunity.

The invention provides a kind of Chinese medicine composition; use standard and conventional technology; the present composition acceptable solid or liquid-carrier on galenic pharmacy are combined, and make it at random on galenic pharmacy acceptable adjuvant and excipient be combined and be prepared into microgranule or microsphere.Solid dosage forms comprises pill, granule, tablet, soft extract, syrup, mixture, drop pill, capsule, medicated wine, tincture etc.Solid carrier can be at least one material, and it can serve as diluent, flavouring agent, solubilizing agent, lubricant, suspending agent, binding agent, disintegrating agent and coating agent.Inert solid carrier comprises magnesium phosphate, magnesium stearate, smoothers sugar, lactose, pectin, propylene glycol, polyoxyethylene sorbitan monoleate, dextrin, starch, gelatin, cellulose substances such as methylcellulose, microcrystalline Cellulose, low melt point paraffin, Polyethylene Glycol, mannitol, cocoa butter etc.

The present invention about the more detailed description of compositions and technical study as follows:

One, formula for a product (1000)

Radix Panacis Quinquefolii 187.5g cervus elaphus linnaeus 37.5g Radix Rhodiolae 375g Fructus Lycii 375g Herba Dendrobii 125g

Two, raw materials used efficacy effect, consumption and each material combination relation

(1) raw materials used efficacy effect and consumption (for tablet)

the raw materials used consumption of table 1

Effect of medical material of the present invention is as follows:

Araliaceae Radix Panacis Quinquefolii panaxquinque folium.Ldry root.Having another name called Radix Panacis Quinquefolii, is nourishing one of rare medicinal herbs, sweet, the micro-hardship of its nature and flavor, cool.There is effect of boosting qi and nourishing yin, clearing away heat and promoting production of body fluid.Clinically be used for physical strength reinforcing, anticancer growth, increase immunologic function, regulate blood pressure, strengthening cardiac muscle and strengthen the mobility, hypermnesis ability etc. of heart.Radix Panacis Quinquefolii chemical composition mainly contains saponin, polysaccharide, aminoacid and volatile oil, fatty acid, trace element, vitamin, irony, the multiple nutrients material such as calcareous.

Animal in deer family Cervus elaphus linnaeus cervus elaphusthe young horn of the unossified close raw fine hair of stag of Linnaeus.Its nature and flavor are sweet, salty, temperature.Return kidney, Liver Channel.There is invigorating kidney-YANG, benefiting essence-blood, bone and muscle strengthening, adjust punching to appoint, effect of holder sore.Chemical composition more complicated in cervus elaphus linnaeus, comprises organic principle and inorganic constituents.Organic principle includes 19 kinds with upper amino acid, 9 kinds of fatty acids, 10 kinds of phospholipid compositions, wherein obviously has the component of pharmacologically active to be protein and peptide, steroid and polyamine compounds.

Radix Rhodiolae rhodiola crenulata(Hook.f.etThoms.) the dry root welding technology of H.Ohba.Nature and flavor are sweet, bitter, flat.There is benefiting QI for activating blood circulation, effect of relievining asthma of promoting blood circulation., apoplectic hemiplegia, asthenia asthma pained for blood stasis due to qi deficiency, breast fraud.Radix Rhodiolae applicating history is long, and before more than 2,000 year, the people of Qinghai-Tibet Platean is used as medicine with it, for building body, and the impact of opposing poor environment.Among the people being commonly used to is decocted water or steeps in wine, and to banish fatigue, goes back preventable disease simultaneously and to be healthy and strong and nourishing lengthens one's life.Because it has magical effect of strengthening healthy qi, benefiting qi and nourishing blood, YIN nourishing lung benefiting, successive dynasties Tibetan medicine is regarded as " lucky three essentials-essence ".Its main chemical has rhodioside, butyl alcohol and Radix Rhodiolae polysaccharide, and containing flavonoid, protein, fat and trace element, aminoacid etc.

Lycium barbarum lycium barbaruml. dry mature fruit.Nature and flavor are sweet, flat.Return liver, kidney channel.There is effect of nourishing the liver and kidney, replenishing vital essence to improve eyesight.For asthenia damage of essence, soreness of waist and knee joint, vertigo and tinnitus, the disease such as impotence and seminal emission, interior-heat are quenched one's thirst, blood deficiency and yellow complexion, blurred vision are not clear.Fructus Lycii has many-sided pharmacological action and bioactive functions.Containing multiple nutritional components and trace element in Fructus Lycii, its principle active component lycium barbarum polysaccharide (LBP), has the effects such as resisting fatigue, radioprotective, enhancing immunity.

Herba Dendrobii ( dendrobium officinalekimura et Migo) cultivate the dry stem of product.Sweet in the mouth, is slightly cold.Return stomach, kidney channel.There is reinforcing stomach reg fluid, the effect of nourishing YIN and clearing away heat.Lose for cloudy impairment of body fluid, xerostomia excessive thirst, deficiency of stomach-Yin, lack of appetite is retched, and deficiency-heat is not moved back after being ill, hyperactivity of fire caused by deficiency of YIN, hectic fever due to YIN-deficiency consumptive fever, and poor vision, muscles and bones is withered soft.Modern pharmacological research shows, Herba Dendrobii has aspect pharmacological actions such as strengthening immunity, resisting fatigue, antioxidation, digestion promoting, promotion salivation, blood sugar lowering, blood pressure lowering, anti-liver injury, antitumor.Herba Dendrobii is mainly containing compounds such as polysaccharide, alkaloid, luxuriant and rich with fragrance compounds, amino acidses.

Five tastes raw material and Radix Panacis Quinquefolii, cervus elaphus linnaeus, Radix Rhodiolae, Fructus Lycii, Herba Dendrobii in this product formula.Sweet, the micro-hardship of Radix Panacis Quinquefolii, cool, there is boosting qi and nourishing yin, clearing away heat and promoting production of body fluid effect; Cervus elaphus linnaeus is sweet, salty, and temperature, can invigorating kidney-YANG, benefiting essence-blood, bone and muscle strengthening; Radix Rhodiolae is sweet, bitter, flat, can benefiting QI for activating blood circulation, promotes blood circulation and relievings asthma; Fructus Lycii is sweet, and flat, have flat invigorating the liver and kidney, replenishing vital essence to improve eyesight effect, Herba Dendrobii is sweet, is slightly cold, and has reinforcing stomach reg fluid, effect of nourishing YIN and clearing away heat.

(2) dominating process route

This product through pretreatment, extraction, concentrated, dry, sieve, mix, granulate, drying, granulate, always the main technique such as to mix and make.

(3) technique definition base

1) Radix Rhodiolae, Fructus Lycii and Herba Dendrobii abstract methods is preferred

With crude polysaccharides, rhodioside for inspection target, using extraction time, amount of water, extraction time as influence factor, design Three factors-levels orthogonal test, factor level is in table 2:

table 2 extracts orthogonal test factor level table

Test method and result: take Radix Rhodiolae 375g, Fructus Lycii 375g, Herba Dendrobii coarse powder 125g totally nine parts, every part of 875g, extracts respectively, filter, merging filtrate, concentrating under reduced pressure (70 ~ 80 DEG C,-0.06 ~-0.1Mpa), drying under reduced pressure, measure crude polysaccharides, Determination of Salidroside.According to table arranges test, and test arrangement and the results of analysis of variance are in table 3,4,5.

the analysis of variance table that table 4 is index with crude polysaccharides content

Take crude polysaccharides as the results of analysis of variance display of index, the extraction of extraction time on crude polysaccharides has appreciable impact (P < 0.05), amount of water and extraction time are on the extraction impact of crude polysaccharides not significantly (P > 0.05), in conjunction with intuitive analysis result, determine that the optimal level of crude polysaccharides is combined as A ₃b ₃c ₃.

Take rhodioside as the results of analysis of variance display of index, the extraction of extraction time on rhodioside has appreciable impact (P < 0.05), amount of water and the extraction of extraction time to rhodioside do not make significant difference (P > 0.05), in conjunction with intuitive analysis result, determine that the optimal level of rhodioside is combined as A ₂b ₃c ₃.

Comprehensive the above results, the extraction of extraction time crude polysaccharides and rhodioside has appreciable impact, known in conjunction with intuitive analysis, crude polysaccharides and rhodioside extract 2 times and extract 3 times, extraction effect is more or less the same, therefore from the cost-saving consideration of actual production, and selective extraction 2 times; The extraction impact of amount of water on crude polysaccharides and rhodioside is not remarkable, from the cost-saving consideration of actual production, chooses 8 times amount that add water; The extraction impact of extraction time on crude polysaccharides and rhodioside is all not remarkable, and in conjunction with intuitive analysis, excellent level is C ₃.Therefore optimum extraction process is decided to be A ₂b ₂c ₃, namely add water 8 times amount, extracts 2 times, and each 2 hours is optimised process.

2) demonstration test

To the condition A optimized ₂b ₂c ₃carry out demonstration test, concrete operations are as follows:

Take Radix Rhodiolae 375g, Fructus Lycii 375g, Herba Dendrobii coarse powder 125g totally three parts, extract respectively, filter, merging filtrate, concentrating under reduced pressure (70 ~ 80 DEG C ,-0.06 ~-0.1Mpa), drying under reduced pressure, measure crude polysaccharides and Determination of Salidroside.The results are shown in Table 6.

table 6 demonstration test table

As can be seen from demonstration test: average paste-forming rate is 25.2%, and the average extracted amount of crude polysaccharides is 16.51g, and the average extracted amount of rhodioside is 1.44g, and extraction process is stablized, feasible.

to sum up result of study, determine that extraction process is:

Get formula ratio Herba Dendrobii, Fructus Lycii, Radix Rhodiolae add 8 times amount soak by water 2 times, each 2 hours, filter, merging filtrate, filtrate reduced in volume (70 ~ 80 DEG C ,-0.06 ~-0.1Mpa) is to the extractum of relative density 1.05 ~ 1.15 (60 DEG C); Get extractum and carry out spraying dry (inlet temperature is 170-180 DEG C, and leaving air temp is 95-100 DEG C), cross 80 mesh sieves, to obtain final product.

three, reach core hall board and reach the report of core sheet alleviating physical fatigue function animal experiment

l materials and methods

1.1 laboratory animal

SPF level Male Kunming strain mice, 18-22g, 160.Mice and mouse feed are provided by Animal Experimental Study center, Hubei Province, and laboratory animal and Feed Manufacturing credit number are SCXK(Hubei Province) 2008-0005, laboratory animal occupancy permit number is SYXK(Hubei Province) 2012-0065.Animal feeding room temperature is 20 ~ 25 DEG C, and humidity is 40 ~ 70%.

1.2 sample sources and process

Reach core hall board to reach core sheet and provided by Tianjin Zhongxin Pharmaceutical Group Co, crowd recommends daily intaking amount to be 6.592g/60kg BW.The function sample that censorship unit provides is the raw mixture removing adjuvant, and it is 54.60% that function sample accounts for finished product ratio, and function sample crowd recommends daily intaking amount to be 3.599g/60kg BW (i.e. 0.060/kg BW).

Sample preparation: accurately take test sample 6.Og, 12.Og, 18.Og, adding distil water dilutes and stirs and is settled to 200mL, then tested material concentration is respectively 0.03g/ml, 0.06g/ml, 0.09g/ml, and use as basic, normal, high dosage group gavage, negative control group gives distilled water.

1.3 dosage groupings

Calculating by the function sample of censorship recommends consumption to be 3.599g/60kg BW(and 0.060/kg BW crowd's day), as dose design foundation, design 0.6,1.2,10,20,30 times of 1.8g/kg BW(respectively suitable crowd's recommended amounts) as three dosage groups, establish negative control (distilled water) group simultaneously.Establish 4 experimental grouies altogether, often organize 40.Gavage capacity is 20ml/kgBW.Test is started after 30 days.Test one group of swimming with a load attached to the body experiment, test two groups of blood lactase acid experiments, test three groups of serum urea experiments, test four groups of hepatic glycogen content experiments.

1.4 experimental technique

1.4.1 Loaned swimming test

Method: the continuous gavage of mice 30 days, last is to 30min after tested material, and mouse tail root bears a heavy burden 5% body weight, puts swimming trunk went swimming and observes mice from swimming to the death time.Room temperature: 24-26 DEG C, water temperature: 25 DEG C ± 1 DEG C.

1.4.2 the mensuration of Serum lactic acid content

Method: the continuous gavage of mice 30 days, last, to 30min after tested material, puts into the water went swimming 10min of 30 DEG C, before swimming, after swimming at once, swimming have a rest after 20 min get blood (endocanthion gets blood) respectively for three times and measure blood lactase acid value.Used kit builds up Bioengineering Research Institute by Nanjing and provides, test kit lot number: 20140409.Room temperature: 24-26 DEG C.

1.4.3 the mensuration of serum urea content

Method: the continuous gavage of mice 30 days, last, to 30min after tested material, puts into the water went swimming 90min of 30 DEG C, and after water outlet, rest 60min, eyeball gets hematometry serum urea content.Serum urea measures and adopts Hitachi 7020 type automatic clinical chemistry analyzer to measure, and used kit is provided by Fenghui Medical Science and Technology Co., Ltd., Shanghai, test kit lot number: 140524.Room temperature: 24-26 DEG C.

1.4.4 the mensuration of hepatic glycogen content

Method: the continuous gavage of mice 30 days, last, to 30min after tested material, is got liver and is measured hepatic glycogen content.Used kit builds up Bioengineering Research Institute by Nanjing and provides, test kit lot number: 20140409.Room temperature: 24-26 DEG C.

1.5 statistical analysis

Adopt variance analysis to add up, but first need carry out homogeneity test of variance by the program of variance analysis, variance is neat, calculates F value, F value < FO.05, conclusion: no significant difference between each group mean; F value >=FO.05, P≤0.05, adds up with the comparative approach between two of mean between multiple experimental group and a matched group; Suitable variable transitions is carried out to the data of abnormal or heterogeneity of variance, after meeting normal state or variance and requiring together, adds up by the data after conversion; If do not reach normal state or the neat object of variance after variable transitions yet, use rank test instead and add up.

2 results

2.1 combined extracts are on the impact of Mouse Weight

combined extractsincrease Mouse Weight and have no significant effect, the forward and backward body weight of each test group mouse test compares P>O.05(in table 7,8,9,10 with negative control group).

2.2 combined extracts are on the impact of mice burden swimming time

combined extractshigh dose group can extend the mice burden swimming time, and comparing with negative control group has significant difference

(P<0.05), in table 11.

2.3 combined extracts are on mice swimming blood lactase acid value and the impact of blood lactase acid area under curve

Compare with negative control group, reach core hall board and reach the rear blood lactase acid value (P<0.05) of core sheet middle and high dosage group energy significance reduction mice swimming, high dose group energy significance reduces the blood lactase acid value (P<0.05) after having a rest, in table 13, table 14; Compare with negative control group, middle and high dosage energy significance reduces the blood lactase acid area under curve (P<0.05) of mice swimming, in table 15; And each dosage group changes (p>0.05) to blood lactase acid value before swimming without significance, in table 12.

2.4 combined extracts are on the impact of mice serum carbamide

Reach core hall board to reach the middle and high dosage group of core sheet and significantly can reduce mice serum carbamide value, compare with negative control group and have significant difference (P<0.05), in table 16.

2.5 combined extracts are on the impact of Mouse Liver glycogen content

Reach core hall board reach each dosage group of core sheet on Mouse Liver glycogen content impact compare with negative control group, difference that there are no significant (P>0.05), in table 17.

3 conclusions

Tested material finished product crowd recommends daily intaking amount to be 6.592g/60kg BW, the function sample provided due to censorship unit is the raw mixture removing adjuvant, it is 54.60% that function sample accounts for finished product ratio, therefore function sample crowd recommends daily intaking amount to be 3.599g/60kg BW (i.e. 0.060/kg BW), daily intaking amount function sample crowd is recommended to expand 10,20,30 times of designs, three dosage groups respectively, 0.6 namely, 1.2,1.8g/kg BW, establish negative control group simultaneously.Adopt SPF level Kunming mouse, continuous gavage gives, and start test after 30 days, experimental result is judged as significant difference with P<O.05, and each dosage group compares with negative control group, and result shows:

1) each dosage group is to Mouse Weight there are no significant difference;

2) high dose group can extend the mice burden swimming time;

3) each dosage group affects without significance blood lactase acid value before mice swimming;

4) after middle and high dosage group can reduce mice swimming, blood lactase acid value, high dose group can reduce the rear blood lactase acid value of mice rest;

5) middle and high dosage group can reduce the blood lactase acid area under curve after swimming;

6) middle and high dosage group can reduce mice serum carbamide value;

7) each dosage group on Mouse Liver glycogen content there are no significant impact.

According to alleviating physical fatigue function assessment process regulation, comprehensive above every result of the test judges, reaches core hall board and reaches core sheet and have alleviating physical fatigue function.

four, combined extracts enhancing immunity function animal experiment report

1 materials and methods

1.1 sample sources and process

Reach core hall board to reach core sheet and provided by Tianjin Zhongxin Pharmaceutical Group Co, the raw mixture crowd removing adjuvant recommends daily intaking amount 3.599g/60kg BW (i.e. 0.060g/kg BW).

Sample preparation: accurately measure given the test agent 6.Og, 12.Og, 18.Og, is settled to 200mL with distilled water respectively, for basic, normal, high dosage group mouse stomach.Negative control group gives distilled water.

1.2 laboratory animals and environment

SPF level Kunming kind female mice, 18-22g, 200, mice and mouse feed provide by Animal Experimental Study center, Hubei Province.Laboratory animal and Feed Manufacturing credit number: SCXK(Hubei Province) 2008-0005; Laboratory animal occupancy permit number: SYXK(Hubei Province) 2012-0065.Animal feeding room temperature is 20-25 DEG C, and humidity is 40-70%.

1.3 dosage groupings

Recommend daily intaking amount 0.060g/kg BW to expand 10,20,30 times by crowd and basic, normal, high three dosage groups are set, i.e. 0.60g/kg BW, 1.20g/kg BW, 1.80g/kg BW, be divided into five experimental grouies, every experimental group comprises negative control group, basic, normal, high dosage group, and each dosage group laboratory animal number is 10.Immunization experiment one group carries out delayed allergy experiment; Immunization experiment two groups carries out mouse lymphocyte transformation experiment and the NK cytoactive detection of ConA induction; Immunization experiment three groups carries out Turnover of Mouse Peritoneal Macrophages and engulfs chicken red blood cell experiment; Immunization experiment four groups carries out mensuration and the antibody-producting cell detection of serum hemolysin; Immunization experiment five groups carries out lymphatic organ/weight ratio pH-value determination pH and carbonic clearance experiment.The continuous gavage of mice starts test after 35 days.Each experimental mice all gives by 0.20ml/lOg BW capacity per os gavage.

1.4 test method

1.4.1 the mouse spleen lymphocyte conversion test of ConA induction

Method: asepticly get spleen, is placed in and fills appropriate aseptic Hank's liquid plate, ground by spleen gently, make individual cells suspension with tweezers.Filter through 200 eye mesh screens, wash 2 times with Hank's liquid, each centrifugal lOmin (lOOOr/min).Then by cell suspension in the complete culture solution of 1mL, with the blue dyeing counting viable count of platform phenol (should more than 95%), adjustment cell concentration be 3 × 10 ⁶individual/mL.Add in 24 well culture plates by every a splenocyte suspension point holes, every hole 1mL, a hole adds 75 μ L Con A liquid (being equivalent to 7.5 μ g/mL), and 5%CO in contrast, is put in another hole ₂, 37 DEG C of CO ₂72h is cultivated in incubator.Cultivation terminates front 4h, and every hole sucks supernatant 0.7mL gently, adds 0.7mL not containing the RPMI1640 culture fluid of calf serum, adds MTT (5mg/mL) 50 μ L/ hole simultaneously, continues to cultivate 4h.After cultivation terminates, every hole adds 1mL acid isopropyl alcohol, and piping and druming mixing, makes purple crystal dissolve completely.Then be dispensed in 96 well culture plates, 3 parallel holes (1OO μ L/hole) are made in each hole, measure optical density value by microplate reader with 570nm wavelength.

1.4.2 dinitrofluorobenzene (DNFB) induces delayed allergy (DTH)

Method: ear swelling method.Prepare with the acetone sesame oil solution of 1:1 with 1%DNFB() after sensitized mice, within the 5th day, attack auris dextra with DNFB again, put to death animal after 24h and cut the auricle that left and right auricular concha card punch takes off diameter 8mm, weigh, represent the degree of DTH with the difference of the weight of left and right ear.

1.4.3 antibody-producting cell detects

Get the Sanguis caprae seu ovis of defiber, with brine 3 times, centrifugal (2000r/min) lOmin at every turn, every Mus through lumbar injection 2% (v/v) SRBC O.2mL.The mice cervical dislocation of SRBC immunity after 4 ~ 5 days is put to death, take out spleen, be placed on and be loaded with in the little plate of Hank's liquid, grind spleen gently, make cell suspension, filter through 200 eye mesh screens, centrifugal (1000/min) lOmin, washes 2 times with Hank's liquid, finally by cell suspension in 5mL RPMI1640 culture fluid, counting cells, and cell concentration is adjusted to 5 × 10 ⁶individual/mL.

The mensuration of plaque: after top layer culture medium (lg agarose adds distilled water to lOOmL) heating for dissolving, put into 45 ~ 50 DEG C of water bath heat preservations, mix with the Hank's liquid of equivalent pH7.2 ~ 7.4,2 times of concentration, subpackage small test tube, often pipe 0.5mL, in pipe, add 50 μ L 1O%SRBC(v/v again, use SA buffer), 20 μ L splenocyte suspensions (5 × 10 ⁶individual/mL), rapid mixing, be poured on the slide of own brush agarose thin layer, do parallel plate, after agar solidification, slide level is buckled and is placed on horse, put into CO2 gas incubator and hatch 1.5h, then add in slide frame groove with the complement (1:8) of SA buffer dilution, after continuing incubation 1.5h, counting hemolysis plaque number.

1.4.4 the mensuration of serum hemolysin

Method: Hemagglutination Method.Get Sanguis caprae seu ovis, with brine 3 times, centrifugal (2000r/min) lOmin at every turn.Hematocrit SRBC normal saline is made into the cell suspension of 2% (v/v), every Mus lumbar injection 0.2mL carries out immunity.After 4 ~ 5 days, except eyeball gets blood in centrifuge tube, place about 1h, solidification blood and tube wall are peeled off, serum is fully separated out, the centrifugal lOmin of 2000r/min, collect serum.

Agglutination: with normal saline by serum doubling dilution, dilution for difference serum is placed in Microhemagglutination brassboard respectively, every hole 1OO μ L, add lOO μ L0.5% (v/v) SRBC suspension again, mixing, put into moistening square position and add a cover, in 37 DEG C of incubation 3h, observing hemagglutination degree.Level calculation according to serum cohesion degree goes out antibody product.

1.4.5 mice carbonic clearance is tested

Method: the india ink (lOmL/kg) injecting dilution by body weight from mouse tail vein, treats that prepared Chinese ink injects, and immediately after reckon by time to pour into prepared Chinese ink 2, lOmin, gets blood 20 μ L respectively from angular vein clump, exists side by side to be added to 2mLO.1%Na2CO by it ₃in solution.Each suction 0.1mL in 96 hole ELISA Plate, by microplate reader at 600nm wavelength place densitometric value (OD), with Na2C0 ₃solution makes negative control.

By sacrifice, get liver and spleen, blot organ surface blood stains with filter paper, weigh respectively.

The ability of mice carbonic clearance is represented with phagocytic index.

Be calculated as follows phagocytic index a.The phagocytic index of given the test agent group is significantly higher than matched group, can judge this experimental result positive.

1.4.6 peritoneal macrophage engulfs chicken red blood cell test

Method: half intracorporal method.The chicken erythrocyte suspension of preparation 20%; Every this suspension of Mus lumbar injection 1mL, puts to death animal after 30min, is faced upward position and be fixed on Mus plate, open abdomen, through Intraperitoneal injection normal saline 2mL, rotates Mus plate 1min, then, sucking-off abdominal cavity washing liquid 1mL, average mark drips on 2 microscope slides, 37 DEG C of incubation 30min; Educate to finish and use normal saline rinsing, dry, fix with the acetone methanol solution of 1:1,4%Giemsa-phosphate buffer dyeing 3min, then dry with distilled water rinsing.Counting 100 macrophages under oil mirror, are calculated as follows phagocytic rate and phagocytic index:

1.4.7 NK cytoactive detection

Method: lactic acid dehydrogenase (LDH) algoscopy.

Go down to posterity (the YAC-1 cell) of target cell:

Before experiment, target cell is carried out Secondary Culture by 24h.Washing 3 times with front with Hank's liquid, is 4 × 10 with RPMI1640 complete culture solution adjustment cell concentration ⁵individual/mL.

The preparation (effector lymphocyte) of splenocyte suspension:

Asepticly get spleen, be placed in the little plate filling appropriate aseptic Hank ' s liquid, gently spleen ground with tweezers, make single cell suspension.Filter through 200 eye mesh screens, wash 2 times with Hank's liquid, each centrifugal lOmin (lOOOr/min).Abandon supernatant cytoplasm is upspring, add 0.5mL aquesterilisa 20 seconds, 0.5mL2 times of Hank's liquid and 8mLHanks liquid is added again after splitting erythrocyte, lOOOr/min, lOmin is centrifugal, resuspended containing the RPMI1640 complete culture solution of 10% calf serum with 1mL, with counting after 1% glacial acetic acid dilution (viable count should more than 95%), with the blue dyeing counting viable count of platform phenol (should more than 95%), finally adjusting cell concentration with RPM11640 complete culture solution be 2 × 10 ⁷individual/mL.

NK cytoactive detects:

Get target cell and each 100 μ L of effector lymphocyte (effect target is than 50:1), add in U-shaped 96 well culture plates: target cell Spontaneous release hole adds target cell and culture fluid each lOO μ L, the maximum release aperture of target cell adds target cell and 1%NP40 each 1OO μ L; Above-mentionedly everyly all establish three parallel holes, in 37 DEG C, 5%C0 ₂4h is cultivated in incubator, then by 96 well culture plates with the centrifugal 5min of 1500r/min, every hole is drawn in 96 well culture plates at the bottom of supernatant 1OO μ L horizontalization, add LDH matrix liquid 1OO μ L simultaneously, according to room temperature differential responses 3 ~ lOmin, every hole adds the HCL 30 μ L of 1mol/L, measures optical density value (OD) at microplate reader 490nm place.Be calculated as follows NK cytoactive, the NK cytoactive of given the test agent group is significantly higher than the NK cytoactive of matched group, can judge this experimental result positive.

1.5 statistical method

Use SPSS software, the method compared between two of one factor analysis of variance method and multiple experimental group and a matched group mean, compares dosage group and whether matched group is variant.If any one dosage group and matched group comparing difference have significance and strengthen (P<0.05), then this experiment is positive.

General employing variance analysis, but first need carry out homogeneity test of variance by the program of variance analysis, variance is neat, calculate F value, F value <FO.05, conclusion: no significant difference between each group mean: F value >=FO.05, P≤0.05, adds up with the comparative approach between two of mean between multiple experimental group and a matched group.

Suitable variable transitions is carried out to the data of abnormal or heterogeneity of variance, after meeting normal state or variance and requiring together, adds up by the data after conversion; If do not reach normal state or the neat object of variance after variable transitions yet, use rank test instead and add up.

2 results

2.1 combined extracts are on the impact of Mouse Weight:

Reach core hall board to reach core sheet and have no significant effect Mouse Weight, each dosage group compares P>O.05 with negative control group, in table 18, table 19, table 20, table 21, table 22.

2.2 combined extracts are on the impact of mouse lymph organ/body weight ratio:

Reach core hall board to reach each dosage group of core sheet and all obviously can not strengthen animal lymph organ/body weight ratio, compare with negative control group, P>O.05, in table 23.

the impact that 2.3 combined extracts transform mouse spleen lymphocyte:

Reach core hall board and reach the spleen lymphocyte proliferation ability that each dosage group of core sheet all obviously can not strengthen mice ConA induction, compare P>O.05 with negative control group, in table 24.

2.4 combined extracts are on the impact of mice delayed allergy:

Reach core hall board to reach each dosage group of core sheet and all obviously can not strengthen the DTH reaction that mice brings out DNFB, compare P>O.05 with negative control group, in table 24.

the impact that 2.5 combined extracts detect mouse antibodies cellulation:

Reach core hall board to reach core sheet high dose group and can significantly improve mouse antibodies cellulation quantity, compare with negative control group, P<O.05; In table 25.

2.6 combined extracts are on the impact of mice serum hemolysin titre levels:

Reach core hall board and reach core sheet middle and high dosage group energy significance rising mice serum hemolysin content, compare with negative control group, be P<O.05; In table 26.

2.7 combined extracts are on the impact of mice carbonic clearance function:

Reach core hall board to reach core sheet high dose group and can significantly improve mice carbonic clearance phagocytic index, compare P<O.05 with negative control group; In table 27.

2.8 combined extracts engulf the impact of chicken red blood cell to Turnover of Mouse Peritoneal Macrophages:

Reach core hall board to reach core sheet high dose group and can significantly improve mice phagocytic percentage and mice phagocytic index, compare with negative control group, P<O.05; In table 28.

2.9 combined extracts are on the impact of NK cells in mice activity:

Reach core hall board to reach core sheet high dose group and can obviously strengthen NK cells in mice activity, compare with negative control group, P<O.05, in table 29.

3. conclusion

The raw mixture crowd of the removal adjuvant provided according to censorship unit recommends daily intaking amount 0.060g/kg BW to expand 10,20,30 times and arranges basic, normal, high three dosage groups, namely 0.60g/kg BW, 1.20g/kg BW, 1.80g/kg BW, separately establish negative control group.Adopt SPF level Kunming mouse, continuous gavage gives, and starts to detect related index after 30 days.Experimental result is judged as significant difference with P<O.05, and each dosage group compares with negative control group, and result shows:

1) each dosage group all obviously can not strengthen animal lymph organ/body weight ratio;

2) each dosage group all obviously can not strengthen the spleen lymphocyte proliferation ability that mice ConA induces;

3) each dosage group all obviously can not strengthen the DTH reaction that mice is brought out DNFB;

4) high dose group energy significance improves mouse antibodies cellulation quantity;

5) middle and high dosage group energy significance raises mice serum hemolysin content;

6) high dose group energy significance improves mice carbonic clearance phagocytic index;

7) high dose group energy significance improves Turnover of Mouse Peritoneal Macrophages phagocytic percentage and phagocytic index;

8) high dose group can obviously strengthen NK cells in mice activity;

According to enhancing immunity function assessment process regulation, this tested material has enhancing immunity function.

five, reach core hall board and reach core sheet toxicological test:

Toxicological test sample adopts the given the test agent removing adjuvant, every 0.546g toxicological test sample is equivalent to sizing finished product 1.0g, crowd recommended amounts 6.592g/ people (the 60kg)/sky of sizing finished product is equal to the crowd recommended amounts 3.599g/ people (60kg) of given the test agent/sky, i.e. 0.060g/kg.BW.Experimental result is as follows:

(1) reach core hall board and reach the SPF level Kunming mouse acute oral toxicity test of core sheet to two kinds of sexes, accumulating twice given low reaches after 15.0g/kg BW(is equivalent to this tested material removing adjuvant, adult recommends 250 times of daily intaking amount 0.060g/kg BW), within the observation period of 14 days, animal has no obvious poisoning symptom and death, this product MTD value is greater than 15.0g/kg BW, according to acute toxicity grading evaluation criteria regulation, this given the test agent belongs to nontoxic level;

(2) binomial mutagenicity test (mouse marrow cell micro nuclear test and sperm malformation test) result is feminine gender;

Within (3) 30 days, feeding trial result shows: by given the test agent by 1.50,3.00,6.00g/kg BW dosage is (after being equivalent to this tested material removing adjuvant respectively, adult recommends 25,50,100 times of daily intaking amount 0.060g/kg BW) give 30 days continuously to SPF level Wistar rat, animal has no obvious poisoning symptom and death.Given the test agent each dosage group rat body weight, food-intake, food utilization, hematology, blood biochemical, organ weights, dirty/index such as body ratio and histopathology compare with negative control group, there are no significant for difference, and result is not for find that this given the test agent has obvious toxic action.

Detailed description of the invention

Below in conjunction with embodiment, the present invention is described further, and embodiment is only indicative, never means that it limits the scope of the invention by any way, and the raw material used by the present invention all has commercially available.Wherein:

radix Panacis Quinquefoliipurchased from the precious pharmaceutcal corporation, Ltd of Tianjin Gus, Chinese Medicines&Health Produts limited company produces.

cervus elaphus linnaeuspurchased from Bozhou City Hong Lide Pharmaceutical Co., Ltd, Shuangyang District, Changchun Bo Longlu industry company limited produces.

radix Rhodiolaepurchased from company limited of Tianjin Medical Herb Pieces Factory.

fructus Lyciipurchased from medical material company of Tianjin Zhongxin Pharmaceutical Group Co., Ltd..

herba Dendrobiipurchased from prepared slices of Chinese crude drugs factory of Bright Food Group Yunnan Hongsheng Biological Products Co., Ltd..

Embodiment 1

Radix Panacis Quinquefolii 187.5g

Cervus elaphus linnaeus 37.5g

Radix Rhodiolae 375g

Fructus Lycii 375g

Herba Dendrobii 125g

Microcrystalline Cellulose 80g

Mannitol 250g

Polyvinylpolypyrrolidone 32g

Magnesium stearate 2.4g

Film coating agent 32g.

preparation method:

1.get formula ratio Herba Dendrobii, Fructus Lycii, Radix Rhodiolae add 8 times amount soak by water 2 times, each 2 hours, filter, merging filtrate, filtrate reduced in volume (80 DEG C, 0.1Mpa) is to the extractum of relative density 1.05 ~ 1.15 (60 DEG C);

2.get extractum and carry out spraying dry, cross 80 mesh sieves, get dry extract powder;

3.get above-mentioned dried cream powder, Radix Panacis Quinquefolii powder, cervus elaphus linnaeus powder, mannitol, microcrystalline Cellulose mix 30 minutes, obtain mixed powder;

4.mixed powder adds 60% ethanol soft material, and 18 mesh sieves are granulated; By wet granular 55 DEG C of dry 1h of making to moisture less than 5%, 16 order granulate, obtain dry granule;

5. obtained dry granule adds polyvinylpolypyrrolidone, the magnesium stearate of formula proportion, mixes 15 minutes, obtains always mixed thing; Technology makes oral formulations routinely.

Embodiment 2

Radix Panacis Quinquefolii 150g

Cervus elaphus linnaeus 30 g

Radix Rhodiolae 300g

Fructus Lycii 300g

Herba Dendrobii 100g.

Preparation method:

1. get the Herba Dendrobii of formula proportion, Fructus Lycii, Radix Rhodiolae add 8 times amount soak by water 2 times, each 1 hour, filter, merging filtrate, filtrate concentrates, and is concentrated into the extractum of 60 DEG C of relative densities 1.05;

2. get extractum and carry out spraying dry, sieve, get dry extract powder;

3. get dried cream powder, Radix Panacis Quinquefolii powder, the mixing of cervus elaphus linnaeus powder, obtain mixed powder; Technology makes oral formulations routinely.

Embodiment 3

Radix Panacis Quinquefolii 200g

Cervus elaphus linnaeus 50g

Radix Rhodiolae 400g

Fructus Lycii 400g

Herba Dendrobii 150g.

Preparation method:

1. get the Herba Dendrobii of formula proportion, Fructus Lycii, Radix Rhodiolae add 6 times amount soak by water 3 times, each 1 hour, filter, merging filtrate, filtrate concentrates, and is concentrated into the extractum of 60 DEG C of relative densities 1.10;

2. get extractum and carry out spraying dry, sieve, get dry extract powder;

3. get dried cream powder, Radix Panacis Quinquefolii powder, the mixing of cervus elaphus linnaeus powder, obtain mixed powder; Technology makes oral formulations routinely.

Embodiment 4

Radix Panacis Quinquefolii 180g

Cervus elaphus linnaeus 40g

Radix Rhodiolae 375g

Fructus Lycii 325g

Herba Dendrobii 130g.

Preparation method:

1. get the Herba Dendrobii of formula proportion, Fructus Lycii, Radix Rhodiolae add 10 times amount soak by water 1 time, each 2 hours, filter, merging filtrate, filtrate concentrates, and is concentrated into the extractum of 60 DEG C of relative densities 1.15;

2. get extractum and carry out spraying dry, sieve, get dry extract powder;

3. get dried cream powder, Radix Panacis Quinquefolii powder, the mixing of cervus elaphus linnaeus powder, obtain mixed powder; Technology makes oral formulations routinely.

Claims (7)

1. a Chinese medicine composition for enhancing immunity, is characterized in that it is made up of the raw material of Chinese medicine of following weight:

Radix Panacis Quinquefolii 150-200g

Cervus elaphus linnaeus 30-50g

Radix Rhodiolae 300-400g

Fructus Lycii 300-400g

Herba Dendrobii 100-150g.

2. the Chinese medicine composition of enhancing immunity described in claim 1, is characterized in that it is made up of the raw material of Chinese medicine of following weight:

Radix Panacis Quinquefolii 185-190g

Cervus elaphus linnaeus 35-40g

Radix Rhodiolae 370-380g

Fructus Lycii 370-380g

Herba Dendrobii 120-130g.

3. the Chinese medicine composition of enhancing immunity described in claim 2, is characterized in that it is made up of the raw material of Chinese medicine of following weight:

Radix Panacis Quinquefolii 187.5g

Cervus elaphus linnaeus 37.5g

Radix Rhodiolae 375g

Fructus Lycii 375g

Herba Dendrobii 125g.

4. the Chinese medicine composition of enhancing immunity described in claim 1 or 2 or 3, is characterized in that described compositions refers to oral formulations.

5. the Chinese medicine composition of enhancing immunity described in claim 4, oral formulations wherein refers to pill, granule, tablet, soft extract, syrup, mixture, drop pill, capsule, medicated wine, tincture.

6. the preparation method of the Chinese medicine composition of enhancing immunity described in claim 5, is characterized in that being undertaken by following step:

(1) get the Herba Dendrobii of formula proportion, Fructus Lycii, Radix Rhodiolae add 6 ~ 10 times amount soak by water 1 ~ 3 time, each 1 ~ 2 hour, filter, merging filtrate, filtrate concentrates, and is concentrated into the extractum of 60 DEG C of relative densities 1.05 ~ 1.15;

(2) get extractum and carry out spraying dry, sieve, get dry extract powder;

(3) get dried cream powder, Radix Panacis Quinquefolii powder, the mixing of cervus elaphus linnaeus powder, obtain mixed powder; Technology makes oral formulations routinely.

7. the application of Chinese medicine composition in preparation treatment enhancing immunity, alleviating physical fatigue medicine of enhancing immunity described in claim 1 or 2 or 3.