CN108392543A - A kind of health composition and its preparation method and application - Google Patents
A kind of health composition and its preparation method and application Download PDFInfo
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- CN108392543A CN108392543A CN201810433848.1A CN201810433848A CN108392543A CN 108392543 A CN108392543 A CN 108392543A CN 201810433848 A CN201810433848 A CN 201810433848A CN 108392543 A CN108392543 A CN 108392543A
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Abstract
The present invention relates to field of health care products, more particularly to a kind of mainly to give protection against cancer made of natural medicinal plant, with strengthen immunity, anti-aging, auxiliary, are used for the health composition and its preparation method and application of middle-aged and the old's daily health caring.The health composition is made of the raw material of following parts by weight：5~10 parts of the fruit of Chinese wolfberry；3~8 parts of Radix Astragali；1~6 part of ganoderma lucidum；1~6 part of gynostemma pentaphylla.Health composition of the present invention mainly made of natural medicinal plant, has the function of strengthen immunity, anti-aging, auxiliary anti-cancer etc., can be used for middle-aged and the old's daily health caring.
Description
Technical field
The present invention relates to field of health care products, more particularly to a kind of mainly to exempt from made of natural medicinal plant, with enhancing
Epidemic disease power, anti-aging, auxiliary anti-cancer, are used for the health composition and its preparation method and application of middle-aged and the old's daily health caring.
Background technology
Body immunity is human bioequivalence and eliminates any foreign matter (germ, virus etc.) of external intrusion, processing aging damage
Wound, the own cells to make a variation, and identification and the ability for handling mutant cell and virus infected cell.
Human body is made of 60,000,000,000,000~100,000,000,000,000 cells, there is 500~2000 cell mutations or variation hair daily
Raw, each cell mutates or becomes the different time, and human body robot control system(RCS) can be found in time, and human immune system is ordered to be situated between rapidly
Enter, commander's immune system kills rapidly mutation or mutant, ensures the health status of human body with this.But people is old in entering
Nian Hou, immune function can reduce.
Modern medical science finds that the immunocompetence of human body is a factor for having substantial connection with aging, immune function
Decline is one of most important reason of aging.Some special cells of body immune system can be by the bacterium in invasion body, virus
The cell of aging death, the cell that has been mutated and the substance for causing allergy in vivo, are wholly swallowed and are disappeared
It goes out, makes the stabilization of vivo environment, keep body health.But body's immunity began to decline at 30 years old or so, this variation
Be quietly, slowly, be continued for.
Cell ageing is also a kind of Apoptosis Mechanism, and this mechanism is mainly completed by immune system.Body is by aging
The process of Apoptosis is exactly to avoid the process of cell carcinogenesis, this is just being confirmed in many animals and human research.It removes
Internal various rubbish are immune system most important functions.(gradual aging) becomes not the immune system of human body over time
It is so effective, thus the effect for eliminating senile cell can have a greatly reduced quality.99% disease and immune system disorder it is related (gene,
Except hereditary class disease).
Therefore, the immunity of human body is improved, especially improving the immunity of middle-aged and the old's body seems particularly significant.
Invention content
The purpose of the present invention is to provide a kind of health composition, preparation method and its prepare with strengthen immunity,
It slows down aging, auxiliary preventing cancer function, the application that can be used in the health products of middle-aged and the old's daily health caring.
To achieve the above object, concrete scheme of the invention is as follows：
One of the objects of the present invention is to provide a kind of health composition, the health composition is by following parts by weight
Raw material is made：5~10 parts of the fruit of Chinese wolfberry；3~8 parts of Radix Astragali；1~6 part of ganoderma lucidum；1~6 part of gynostemma pentaphylla.
Preferably, the health composition is made of the raw material of following parts by weight：7 parts of the fruit of Chinese wolfberry；6 parts of Radix Astragali；Ganoderma lucidum 5
Part；5 parts of gynostemma pentaphylla.
The second object of the present invention is to provide a kind of preparation method of health composition, the preparation of the health composition
Method includes the following steps：
A, raw material is weighed by following parts by weight：5~10 parts of the fruit of Chinese wolfberry；3~8 parts of Radix Astragali；1~6 part of ganoderma lucidum；Gynostemma pentaphylla 1
~6 parts；
B, the fruit of Chinese wolfberry is taken to be extracted with water 1~4 time, 0.5~4 hour every time, filtration, merging filtrate, concentration, with 50~
The ethyl alcohol of 98wt% carries out alcohol precipitation separation (alcohol content is 30~80wt%)；Taking precipitate is dried, crushed into fine powder；
C, Radix Astragali is taken to be extracted with water 1~4 time, 0.5~4 hour every time, filtration, merging filtrate was concentrated, is dried, crushed into
Fine powder；
D, ganoderma lucidum is taken to be extracted 1~4 time with the alcohol reflux of 30~98wt%, 0.5~4 hour every time, filtration merged filter
Liquid, recycles ethyl alcohol, and concentration obtains ethanol extract；The dregs of a decoction are extracted with water 1~4 time, and 0.5~4 hour every time, filtration merged filter
Liquid, concentration, obtains water extract；Two kinds of extracts are mixed, fine powder is dried, crushed into；
E, gynostemma pentaphylla is taken to be extracted with water 1~4 time, 0.5~4 hour every time, filtration, merging filtrate, concentration, with 50~
The ethyl alcohol of 98wt% carries out alcohol precipitation separation (alcohol content is 30~80wt%)；It takes filtrate to concentrate, is dried, crushed into fine powder；
F, above-mentioned four kinds of fine powders are uniformly mixed and preparation is made.
Preferably, 7 parts of the fruit of Chinese wolfberry in step A；6 parts of Radix Astragali；5 parts of ganoderma lucidum；5 parts of gynostemma pentaphylla；
Preferably, the extraction time of the fruit of Chinese wolfberry is 2 times in step B；Extraction time is 1.5 hours for the first time, and 1 is small for the second time
When；Alcohol precipitation alcohol content is 70wt%；
Preferably, the extraction time of Radix Astragali is 2 times in step C, and extraction time is 1.5 hours for the first time, and 1 is small for the second time
When；
Preferably, the extraction time of the gynostemma pentaphylla in step D is 2 times, and extraction time is 1.5 hours for the first time, second 1
Hour.
After the fruit of Chinese wolfberry is extracted with water the present invention, then with the method for alcohol precipitation effective ingredient is extracted, is effectively removed without effect
The impurity of effect.
Ganoderma lucidum is first carried out refluxing extraction by the present invention with ethyl alcohol, is then extracted again with water, in this way can will be in ganoderma lucidum
Fat-soluble and water-soluble active ingredient extract as far as possible, it is ensured that the effect of product.In the conditions of the invention, ganoderma lucidum
Active constituent recovery rate highest, and energy expenditure used in reflux course is also minimum.
The third object of the present invention is to provide a kind of health composition application, and the application of health composition refers to preparing
Application in the health products that strengthen immunity, anti-aging, auxiliary give protection against cancer.
Further, the health composition is used for the daily health caring of the middle-aged and the old.
Further, the health composition is tablet, capsule, granule, pill, pastille, mixture, oral administration solution
Agent, powder, soft extract, syrup.
The health composition of the present invention can be made ordinary preparation, can also be sustained release preparation, controlled release preparation, targeting preparation
And various particulate delivery systems.
The pharmaceutical characteristic of each active constituent of the present invention is as follows：
The fruit of Chinese wolfberry, nature and flavor sweet and neutral, returns liver and kidney channel.Function tonification liver kidney, benefiting shrewd head.It cures mainly, is used for consumption consumptive loss, waist
Knee joint soreness, dizziness and tinnitus, impotence and seminal emission, Heat Diabetes, blood deficiency chlorosis, blurred vision.
《Bencao Jingshu》Described in, the fruit of Chinese wolfberry " specially in kidney tonifying "；
《Explaining abstruseness of the canon of materia medica》Described in, the fruit of Chinese wolfberry " tonifying kidney and benefiting sperm "；
《Dictionary of medicinal plant》Described in, the fruit of Chinese wolfberry " nourshing kidney, moistening lung, tonifying liver ".
Wang Jihui, Li Hongmei, Lycium barbarum polysaccharides immunoregulation effect research [Qiqihar Medical College's journal, 2002,23
(11):1204.], polysaccharides can significantly improve the phagocytic function of phagocyte, improve the proliferative capacity of T lymphocytes, increase
Serum IgG content has the function of to improve body nonspecific immunity and specific immune function.
Liu Yanping, Li Jidong, Lycium barbarum polysaccharides [cure in Qinghai the immunoregulation effect of NK cells in mice and leukocyte activity
Institute's journal, 22 (1)：2001-2002], result of study shows that polysaccharides can enhance NK cell killing activities and increase white thin
Born of the same parents' quantity, the reduction to the NK cell killing activities that cyclophosphamide is induced, Lycium barbarum polysaccharides have return action.Lycium barbarum polysaccharides
With positive immunoregulation effect.
Li Xiaoli etc., influence [Journal of Nutrition 2000,22 (4) of the Lycium barbarum polysaccharides to mouse hypoxia-bearing capability:337-
339], test result shows that Lycium barbarum polysaccharides have the function of improving experimental animal resist oxygen lack.
Radix Astragali, property is sweet, tepor.Return lung, the spleen channel.Function tonifying Qi and lifting yang, strengthening exterior and reducing sweat, inducing diuresis for removing edema, shengjin nourishing, row are stagnant
Logical numbness, pus draining and toxin expelling, expelling pus and promoting granulation.For treatment deficiency of vital energy weak, anorexia and loose stool, the sinking of qi of middle-jiao, rush down prolapse of the anus for a long time, uterine bleeding of having blood in stool, exterior deficiency
Spontaneous perspiration, qi deficiency edema, Heat Diabetes, blood deficiency chlorosis, hemiplegia, numbness pain is numb, ulcer is difficult to burst, burst for a long time and does not holds back.
《It does not record》：" the dirty ailment said due to cold or exposure gas of housewife people's, by extravesated blood between the five internal organs, benefit internal organs are deficient, and the exhaustion or lesion of the five internal organs is thin thin, quenches the thirst, abdominal pain,
Let out dysentery, QI invigorating, Li Yin gas.”
《Pearl sac》：" beneficial stomach Qi goes flesh hot, and only spontaneous perspiration, all pains use it.”
《The Decoction and Material Medica》：" heart cloud：Tonifying five zang organs are all empty insufficient, and rush down yin fire, go abnormal heat.It is lossless, it is sent out, has sweat then to stop
It.”
《Collected statements on the herbal foundation》：" arrest sweating, wind dispelling fortune poison are defended in tonifying lung invigorating the spleen in fact.”
Jing Xuening, astragalus polyose inducing dendritic shape cell maturation and the experimental study (mountain for promoting specificity antineoplastic immunity
Eastern university of TCM's doctoral thesis, in May, 2013), result of study shows that astragalus polyose can be with external evoked DC maturations.Through Huang
The DC vaccines of astragalus polysaccharides induced maturation effectively can offer antigen to T cell, promote proliferation and the activation of T cell, increase anti-swollen
The secretion of carcinoma lytokine activates the ability of CTL specificity antineoplastics, it is shown that good extracorporeal anti-tumor effect.Radix Astragali is more
The DC vaccines of sugar induction can pass through the production of increase tumor-bearing mice T cell proliferative capacity, promotion cytokine TNF-α, IL-12
Raw, induction Th1/Th2 enhances antitumor immunity of organism function to mechanism such as Th1 conversions, slows down the tumor-bearing mice knurl speed of growth,
Play extending life effect.
Xiao Feng etc., astragaloside extract to the protective effect of Renal of Diabetic Rats (CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2005,10:
2014-2018), experimental result illustrates that astragaloside extract has certain Renoprotective Effect to diabetes rat.
Jiang Chenlu etc., astragalus polyose Study immune regulation progress [Food Science, 2013,34 (11)：327-332],
The result shows that Radix Astragali be simply can medicine-food two-purpose traditional Chinese medicine, wherein the most active constituent of content be astragalus polyose.It is yellow
Astragalus polysaccharides are a kind of natural extract matter having multi-efficiency, have and adjust blood glucose, and protection liver kidney adjusts immunity and anti-oxidant
Anti-aging effect is especially the most prominent with immunoloregulation function.
Ganoderma lucidum, property sweet and neutral.The thoughts of returning home, lung, liver kidney channel.Function invigorating qi for tranquilization, it is relieving cough and asthma.For having no peace of mind, the heart of having a sleepless night
Throb with fear, deficiency syndrome of the lung cough and asthma, consumptive disease is losed heart, do not feel like eating.
《This warp》：" god is protected in the main deafness of purple sesame, sharp joint, and strengthening the essence gas, hard muscles and bones, good color, long term usage is made light of one's life by commiting suicide does not prolong always
Year.”
《Detailed outline》：" purple sesame treats consumptive disease.”
Zhang Xinxin, Black Ganoderma polysaccharide antitumor activity and its Molecular Mechanism (University Of Nanchang's doctoral thesis, 2014
May), result of study shows that Black Ganoderma polysaccharide has very strong antitumor and immunoregulation effect in vivo, and mechanism of action can
Can be by activated immune organ, immune cell activated and immune signal access, to improve immunity of organisms；By activating G eggs
White signal Signal Transduction Pathways and mitochondrial apoptosis access finally realize its stronger antitumor work to promote apoptosis of tumor cells
With.
Gynostemma pentaphylla, nature and flavor：Micro-sweet；It is cool in nature.Channel tropism：Return lung；Spleen；Kidney channel.Effect：It replenishes qi to invigorate the spleen, preventing phlegm from forming and stopping coughing, heat-clearing solution
Poison.Effect is classified：Qi-restoratives medicine；Apophlegmatisant；Antipyretic.
It cures mainly：It is physically weak weak；Nephrasthenia emission；Leukopenia；Hyperlipidemia；Virus hepatitis；Chronic gastroenteritis；Slowly
Property tracheitis.
The beneficial effects of the invention are as follows：
1, health composition of the invention has a wide range of application, and has strengthen immunity, anti-aging, auxiliary anti-cancer, reaches
The purpose to prolong life meets the requirement of most of crowds.
2, the fruit of Chinese wolfberry is extracted with water the present invention, then removes impurity with the method for alcohol precipitation, can greatly reduce dose；Ganoderma lucidum
Refluxing extraction, the dregs of a decoction are carried out using ethyl alcohol to put and decocted with water, it, can will be clever with the solvent extraction ganoderma lucidum of two kinds of opposed polarities
The fat-soluble active ingredient and aqueous soluble active constituent of sesame all extract, and substantially increase the functional effect of this product, meanwhile,
Under conditions of the present invention, the utilization rate of raw material also highest；Radix Astragali active ingredient is mainly water soluble ingredient, therefore adopts and be extracted with water
Method extract.The extraction process of this product medicinal material only used the solvent of the safety such as water and ethyl alcohol, and not using any has
The raw material and reagent of poison, product safety are without side-effects.
3, present invention process it is scientific and reasonable, it is of low cost, be suitable for large-scale production.
Specific implementation mode
The present invention is further described by the following embodiment, but embodiments of the present invention are unlimited so.
Embodiment 1
The preparation method of health composition includes the following steps：
A, 1000 grams of the fruit of Chinese wolfberry is weighed；800 grams of Radix Astragali；600 grams of ganoderma lucidum；600 grams of gynostemma pentaphylla.
B, the fruit of Chinese wolfberry is taken to be extracted with water 1~4 time, 0.5~4 hour every time, filtration, merging filtrate, concentration, with 50~
The ethyl alcohol of 98wt% carries out alcohol precipitation separation (alcohol content is 30~80wt%)；Taking precipitate is dried, crushed into fine powder；
C, Radix Astragali is taken to be extracted with water 1~4 time, 0.5~4 hour every time, filtration, merging filtrate was concentrated, is dried, crushed into
Fine powder；
D, ganoderma lucidum is taken to be extracted 1~4 time with the alcohol reflux of 30~98wt%, 0.5~4 hour every time, filtration merged filter
Liquid, recycles ethyl alcohol, and concentration obtains ethanol extract；The dregs of a decoction are extracted with water 1~4 time, and 0.5~4 hour every time, filtration merged filter
Liquid, concentration, obtains water extract.Two kinds of extracts are mixed, fine powder is dried, crushed into；
E, gynostemma pentaphylla is taken to be extracted with water 1~4 time, 0.5~4 hour every time, filtration, merging filtrate, concentration, with 50~
The ethyl alcohol of 98wt% carries out alcohol precipitation separation (alcohol content is 30~80wt%)；It takes filtrate to concentrate, is dried, crushed into fine powder；
By four kinds of powder mixings made from above-mentioned steps, be added right amount of auxiliary materials, it is tabletted to get.
Embodiment 2
A, 700 grams of the fruit of Chinese wolfberry is weighed；600 grams of Radix Astragali；500 grams of ganoderma lucidum；600 grams of gynostemma pentaphylla.
B, the fruit of Chinese wolfberry is taken to be extracted with water 1~4 time, 0.5~4 hour every time, filtration, merging filtrate, concentration, with 50~
The ethyl alcohol of 98wt% carries out alcohol precipitation separation (alcohol content is 30~80wt%)；Taking precipitate is dried, crushed into fine powder；
C, Radix Astragali is taken to be extracted with water 1~4 time, 0.5~4 hour every time, filtration, merging filtrate was concentrated, is dried, crushed into
Fine powder；
D, ganoderma lucidum is taken to be extracted 1~4 time with the alcohol reflux of 30~98wt%, 0.5~4 hour every time, filtration merged filter
Liquid, recycles ethyl alcohol, and concentration obtains ethanol extract；The dregs of a decoction are extracted with water 1~4 time, and 0.5~4 hour every time, filtration merged filter
Liquid, concentration, obtains water extract.Two kinds of extracts are mixed, fine powder is dried, crushed into；
E, gynostemma pentaphylla is taken to be extracted with water 1~4 time, 0.5~4 hour every time, filtration, merging filtrate, concentration, with 50~
The ethyl alcohol of 98wt% carries out alcohol precipitation separation (alcohol content is 30~80wt%)；It takes filtrate to concentrate, is dried, crushed into fine powder；
By four kinds of powder mixings made from above-mentioned steps, be added right amount of auxiliary materials, it is tabletted to get.
Embodiment 3
A, 500 grams of the fruit of Chinese wolfberry is weighed；300 grams of Radix Astragali；100 grams of ganoderma lucidum；100 grams of gynostemma pentaphylla.
B, the fruit of Chinese wolfberry is taken to be extracted with water 1~4 time, 0.5~4 hour every time, filtration, merging filtrate, concentration, with 50~
The ethyl alcohol of 98wt% carries out alcohol precipitation separation (alcohol content is 30~80wt%)；Taking precipitate is dried, crushed into fine powder；
C, Radix Astragali is taken to be extracted with water 1~4 time, 0.5~4 hour every time, filtration, merging filtrate was concentrated, is dried, crushed into
Fine powder；
D, ganoderma lucidum is taken to be extracted 1~4 time with the alcohol reflux of 30~98wt%, 0.5~4 hour every time, filtration merged filter
Liquid, recycles ethyl alcohol, and concentration obtains ethanol extract；The dregs of a decoction are extracted with water 1~4 time, and 0.5~4 hour every time, filtration merged filter
Liquid, concentration, obtains water extract.Two kinds of extracts are mixed, fine powder is dried, crushed into；
E, gynostemma pentaphylla is taken to be extracted with water 1~4 time, 0.5~4 hour every time, filtration, merging filtrate, concentration, with 50~
The ethyl alcohol of 98wt% carries out alcohol precipitation separation (alcohol content is 30~80wt%)；It takes filtrate to concentrate, is dried, crushed into fine powder；
By four kinds of powder mixings made from above-mentioned steps, be added right amount of auxiliary materials, it is tabletted to get.
Based on embodiment 2, using orthogonal experiment method, the matrimony vine in dry cream is measured with UV-VIS spectrophotometry
Sub- polyoses content is evaluation index, preferably the optimum process condition of matrimony vine extraction alcohol precipitation.This experiment is horizontal using three, nine because
Element, to investigate the soaking time of the fruit of Chinese wolfberry in extraction process, amount of water, extraction time, concentration densities, alcohol precipitation separating technology contain
The technological parameters such as alcohol amount, time of repose, to determine optimised process.
1 fruit of Chinese wolfberry of table extracts alcohol precipitation process L27(39) orthogonal test designs table
Foundation《Chinese Pharmacopoeia》Version one in 2015, under fruit of Chinese wolfberry medicinal material assay item in polysaccharides assay method
Measure fruit of Chinese wolfberry dry cream polyoses content.
2 orthogonal test calendar of table and orthogonal experiments
According to table 2 as a result, optimum extraction alcohol precipitation process condition be：For the first time plus 10 times of amounts of water, immersion 30 minutes are extracted
1.5 hours, second plus 10 times of amounts of water, extract 1 hour, are condensed into the thick paste that density is 1.16~1.18,95wt% second is added
Alcohol makes alcohol content reach 70wt%, stands 12 hours, and filtering, filter residue volatilizes ethyl alcohol, dry.
It is the pharmacodynamics test research of health composition of the present invention below：
One, cellular immune function assay
(1) influence of the Qi stilbene piece to mouse immune organ
1 test material
1.1 main agents
(Dongguan City Asia Pharmaceutical Co., Ltd provides Qi stilbene piece, lot number：20160614), Content of Tablet of Levamisole Hydrochloride (Guangdong
South China medicine company Group Co., Ltd, Chinese medicines quasi-word H44020759), NaCl (Tianjin Zhi Yuan chemical reagent Co., Ltd, lot number：
2016102067)；
1.2 drug
Qi stilbene piece, 7.00g crude drugs/g, Qi stilbene piece clinic are 23.1g crude drugs/d (3.85g crude drugs/0.55g/ per consumption per day
Grain, one time 3, two times a day), for adult's weight in terms of 60kg, average dosage is 0.385g crude drugs/kg/d, this test mice
Basic, normal, high three dosage groups are set to 3.85,7.7,15.4g crude drugs/kg/d, above-mentioned dosage be clinical medicine dose 10,
20,40 times (according to the weight).Levamisol：This experiment mice dose design is 25mg/kg.
1.3 animal
KM mouse, male, SPF grades, 18~22g, Nanfang Medical Univ's Experimental Animal Center, the animal quality quality certification：
SCXK (Guangdong) 2013-0002.
1.4 feeding environment：23.0 ± 2.0 DEG C, relative humidity 40-70% of experiment periods animal house environment temperature, rate of ventilation>
15 times/hour ,/12 hours dark of illumination in 12 hours, light and shade alternating, experimental animal use credit number：SYXK (Guangdong) 2012-
1125。
1.5 main equipment
HZT-A1000 electronic balances (Foochow Hua Zhi scientific instrument Co., Ltd)；FA2004B electronic balances (upper Nereid section
Tian Mei scientific instrument Co., Ltd)
2 methods
2.1 groupings and administration
Male KM mouse 60, weight 18-22g is taken, 5 groups, i.e. physiological saline group, left-handed miaow are randomly divided by weight
Basic, normal, high 3 dosage groups of azoles positive drug group, Qi stilbene piece (3.85,7.7,15.4g crude drugs/kg), every group 12.Qi stilbene piece is each
Every animal of dosage group and levamisol positive drug group gives the drug of corresponding dosage daily, and gavage volume is 20mL/kg, raw
Reason brine group then gives isometric physiological saline, successive administration 30d.Mouse is weighed at the end of experiment, and cervical dislocation puts to death mouse
After take spleen, thymus gland, weigh.
Thymus index (mg/10g)=(thymic weight × 1000/ mouse weight) × 10
Index and spleen index (mg/10g)=(spleen weight × 1000/ mouse weight) × 10
2.2 statistical procedures
Data analysis is carried out using statistic software SPSS 21.0, generally uses variance analysis, but need to be by the journey of variance analysis
Sequence first carries out homogeneity test of variance, and variance is neat, calculates F values, F values<F0.05, conclusion：No significant difference between each group mean；F values
≥F0.05, P≤0.05 is counted with the comparative approach two-by-two of mean between multiple experimental groups and a control group；To abnormal
Or the data of heterogeneity of variance carry out variable conversion appropriate, after meeting normal state or variance requires together, with transformed data into
Row statistics；If being still not up to normal state or the neat purpose of variance after variable conversion, uses rank sum test instead and counted.
3 results
By table 3 as it can be seen that compared with physiological saline group, the chest gland weight and thymus gland of each dosage group of Qi stilbene piece and levamisol group refer to
The significant differences (P ＜ 0.05 or P ＜ 0.01) of number, Qi stilbene piece is low, spleen weight and spleen of high dose group and levamisol group
Index has certain ascendant trend, but there was no significant difference, and Qi stilbene piece is prompted to have certain enhancing to mouse immune organ weight
Effect.
Influence of the 3 Qi stilbene piece of table to mouse immune organ
Note：Compared with physiological saline group,*P ＜ 0.05,**P ＜ 0.01
4 brief summaries
Mouse continuously gives Qi stilbene piece gavage 30 days, and to mouse immune, organ weight has a certain impact.As a result Qi is prompted
Stilbene piece plays the role of centainly increasing the immune organ weight of mouse.
(2) influence of the Qi stilbene piece to the mouse ConA Splenic vein hemodynamics induced
1 test material
1.1 main agents
(Dongguan City Asia Pharmaceutical Co., Ltd provides Qi stilbene piece, lot number：20160614), Content of Tablet of Levamisole Hydrochloride (Guangdong
South China medicine company Group Co., Ltd, Chinese medicines quasi-word H44020759), NaCl (Tianjin Zhi Yuan chemical reagent Co., Ltd, lot number：
2016102067), RPMI1640 culture solutions (Gibco, lot number：8116157), (Zhejiang day Hangzhoupro biotechnology is limited for calf serum
Company, lot number：20150914), 2 mercapto ethanol (Genview, lot number：6526010150), dual anti-(Solarbio, lot number：
20160509), concanavalin A (Sigma, lot number：201600402), hydrochloric acid (Tianjin great Mao chemical reagent works, lot number：20160715)、
Isopropanol (Tianjin great Mao chemical reagent works, lot number：20140304), MTT (Sigma, lot number：201603000), Hank ' s liquid
(Genview, lot number：6706010110), PBS buffer solution (Hyclone, lot number：ABA2112280), (Guangzhou Xiang is rich for trypan blue
Bio tech ltd, lot number：0108XB15).
1.2 drug
Qi stilbene piece, 7.00g crude drugs/g, Qi stilbene piece clinic are 23.1g crude drugs/d (3.85g crude drugs/0.55g/ per consumption per day
Grain, one time 3, two times a day), for adult's weight in terms of 60kg, average dosage is 0.385g crude drugs/kg/d, this test mice
Basic, normal, high three dosage groups are set to 3.85,7.7,15.4g crude drugs/kg/d, above-mentioned dosage be clinical medicine dose 10,
20,40 times (according to the weight).Levamisol：This experiment mice dose design is 25mg/kg.
1.3 animal
KM mouse, SPF grades, 18~22g, Nanfang Medical Univ's Experimental Animal Center, the animal quality quality certification：SCXK (Guangdong)
2013-0002。
1.4 feeding environment：23.0 ± 2.0 DEG C, relative humidity 40-70% of experiment periods animal house environment temperature, rate of ventilation>
15 times/hour ,/12 hours dark of illumination in 12 hours, light and shade alternating, experimental animal use credit number：SYXK (Guangdong) 2012-
1125。
1.5 main equipment
HZT-A1000 electronic balances (Foochow Hua Zhi scientific instrument Co., Ltd)；FA2004B electronic balances (upper Nereid section
Tian Mei scientific instrument Co., Ltd)；Tecan Infinite 200PRO multi-function microplate readers (Tecan Austria GmbH)；
Pulsation vacuum sterilizer (Xinhua Medical Apparatus Co., Ltd. Shandong)；FA2104 upper ware electronic balances (Shanghai exact science
Instrument Ltd.)；SW-CJ-1F clean benches (SuZhou Antai Air Tech Co., Ltd. of Su Jing groups)；Table-type low-speed is big
Capacity centrifugation machine L550 (Hunan Xiang Yi Laboratory Instruments development corporation, Ltd.)；Blood counting chamber (Shanghai City refinement biochemical reagents
Instrument Ltd.)；BA310-T biomicroscopes (Motic)；3100 carbon dioxide incubators (Thermo).
2 methods
2.1 main solutions are prepared
Complete medium：RPMI1640 culture solutions, with 10% calf serum of preceding addition, 1% glutamine (200mmol/
L), 1% dual anti-(10,000 U/mL) and 5 × 10-5The 2 mercapto ethanol of mol/L, with the HCl or 1mol/L of sterile 1mol/L
NaOH tune pH to 7.0-7.2, i.e. complete culture solution.
ConA liquid：The solution of 100 μ g/mL, filtration sterilization, -20 DEG C of preservations are configured to distilled water.
Sterile Hank ' s liquid：With preceding with 3.5% sterile NaHCO3Adjust pH7.2-7.4.
MTT liquid：5mg MTT are dissolved in the PBS of 1mL pH7.2, it is now with the current.
Acid isopropyl alcoholic solution：The HCl of 4mL 1mol/L, prepared before use are added in 96mL isopropanols.
2.2 groupings and administration
Male KM mouse 60, weight 18-22g is taken, 5 groups, i.e. physiological saline group, left-handed miaow are randomly divided by weight
Basic, normal, high 3 dosage groups of azoles positive drug group, Qi stilbene piece (3.85,7.7,15.4g crude drugs/kg), every group 12.Qi stilbene piece is each
Every animal of dosage group and levamisol positive drug group gives the drug of corresponding dosage daily, and gavage volume is 20mL/kg, raw
Reason brine group then gives isometric physiological saline, successive administration 30d.
2.3MTT method
2.3.1 the preparation of splenocyte suspension
1h after the last administration weighs, sterile to take spleen, thymus gland, weighs, and spleen is placed in and fills sterile Hank ' the s liquid plates of 3mL
In, and one piece of gauze is placed above in spleen, gently spleen is ground with large syringe inner core, single splenocyte suspension is made.Through
200 mesh screens filter, and are rinsed twice with Hank ' s liquid, centrifuge 10min (1000r/min) every time.Then splenocyte is resuspended in
In the complete culture solution of 3mL, with Trypan Blue living cell counting number (viable count answers 95% or more), adjustment cell concentration is
3×106A/mL.
2.3.2 lymphproliferation response
Holes will be divided to be added in 24 well culture plates per a splenocyte suspension, per hole 1mL, 75 μ L ConA are added in a hole
Liquid (is equivalent to 7.5 μ g/mL), and 5%CO as a contrast, is set in another hole272h is cultivated in incubator.Complete medium when 48h
A not good liquor is changed, culture terminates preceding 4h, and supernatant 0.7mL is gently sucked per hole, adds 0.7mL and is free of calf serum
RPMI1640 culture solutions, while 50 holes μ L/ MTT (5mg/mL) are added, mixing is gently blown and beaten, continues to cultivate 4h.After culture,
1mL acid isopropyl alcohol is added per hole, manually blows and beats mixing, purple crystal is made to be completely dissolved, be then dispensed into 96 well culture plates,
Each hole dispenses 3 holes and measures OD value with microplate reader with 570nm wavelength as Duplicate Samples.With the OD value for adding the holes ConA
It subtracts and is not added with the OD value in the holes ConA and represents the proliferative capacity of lymphocyte.
2.4 statistical procedures
Data analysis is carried out using statistic software SPSS 21.0, generally uses variance analysis, but need to be by the journey of variance analysis
Sequence first carries out homogeneity test of variance, and variance is neat, calculates F values, F values<F0.05, conclusion：No significant difference between each group mean；F values
≥F0.05, P≤0.05 is counted with the comparative approach two-by-two of mean between multiple experimental groups and a control group；To abnormal
Or the data of heterogeneity of variance carry out variable conversion appropriate, after meeting normal state or variance requires together, with transformed data into
Row statistics；If being still not up to normal state or the neat purpose of variance after variable conversion, uses rank sum test instead and counted.
3 results
By table 4 as it can be seen that compared with physiological saline group, Qi stilbene piece is low, middle dose group is to the spleen lymphocyte proliferation of mouse
There are humidification, and significant difference (P ＜ 0.01), the spleen lymphocyte proliferation of Qi stilbene piece high dose group and levamisol group
There are certain ascendant trend, but no difference of science of statistics, prompts Qi stilbene piece that there are the mouse ConA Splenic vein hemodynamics induced
Apparent facilitation.
Influence of the 4 Qi stilbene piece of table to mice spleen lymphocytes proliferation ability
Note：Compared with physiological saline group,*P ＜ 0.05,**P ＜ 0.01
4 brief summaries
Mouse continuously gives Qi stilbene piece gavage 30 days, has significantly to the spleen lymphocyte proliferation ability of mouse ConA inductions
Facilitation and the significant meaning of difference.
(3) influence (ear swelling method) of the Qi stilbene piece to mouse delayed allergy (DTH)
1 test material
1.1 main agents
(Dongguan City Asia Pharmaceutical Co., Ltd provides Qi stilbene piece, lot number：20160614), Content of Tablet of Levamisole Hydrochloride (Guangdong
South China medicine company Group Co., Ltd, Chinese medicines quasi-word H44020759), 2,4-dinitrofluorobenzene (the limited public affairs of Chengdu Ai Ke chemical technologies
Department, product batch number：201602291)；Acetone (Kai Xin chemical reagents Co., Ltd of Hengyang City, product batch number：20150807).
1.2 drug
Qi stilbene piece, 7.00g crude drugs/g, Qi stilbene piece clinic are 23.1g crude drugs/d (3.85g crude drugs/0.55g/ per consumption per day
Grain, one time 3, two times a day), for adult's weight in terms of 60kg, average dosage is 0.385g crude drugs/kg/d, this test mice
Basic, normal, high three dosage groups are set to 3.85,7.7,15.4g crude drugs/kg/d, above-mentioned dosage be clinical medicine dose 10,
20,40 times (according to the weight), levamisol：This experiment mice dose design is 25mg/kg.
1.3 animal
KM mouse, male, SPF grades, 18~22g, Nanfang Medical Univ's Experimental Animal Center, the animal quality quality certification：
SCXK (Guangdong) 2013-0002.
1.4 feeding environment：23.0 ± 2.0 DEG C, relative humidity 40-70% of experiment periods animal house environment temperature, rate of ventilation>
15 times/hour ,/12 hours dark of illumination in 12 hours, light and shade alternating, experimental animal use credit number：SYXK (Guangdong) 2012-
1125。
1.5 main equipment
Card punch, FA2004B electronic balances (Shanghai Techcomp Jingke Science Instrument Co., Ltd.).
2 methods
2.1DNFB solution is prepared
DNFB solution answers Fresh, weighs DNFB 50mg, sets in the dry bottle of cleaning, the 5mL acetone that will be prepared in advance
Sesame oil solution (acetone:Sesame oil=1:1) bottle, is poured into, the sealing of bottle stopper blend compounds cloth is covered.It is spare after mixing.
2.2 groupings and administration
Male KM mouse 60, weight 18-22g is taken, 5 groups, i.e. physiological saline group, left-handed miaow are randomly divided by weight
Basic, normal, high 3 dosage groups of azoles positive drug group, Qi stilbene piece (3.85,7.7,15.4g crude drugs/kg), every group 12.Qi stilbene piece is each
Every animal of dosage group and levamisol positive drug group gives the drug of corresponding dosage daily, and gavage volume is 20mL/kg, raw
Reason brine group then gives isometric physiological saline, successive administration 30d.
2.3 sensitization
It is administered the 13rd day, first by mouse part skin cropping, then is lost hair or feathers with depilatory cream, range 3cm × 3cm, in secondary daily
DNFB solution uniformly smears sensitization, every 50 μ L.
The generation of 2.4DTH and measurement
After sensitization 5 days, uniformly it is applied to mouse right ear (two sides) with DNFB solution and is attacked, every 10 μ L.After attack
Cervical dislocation puts to death mouse for 24 hours, cuts left and right auricular concha.The auricle that diameter 8mm is removed with card punch, weighs.With left and right ear weight
Difference indicate DTH degree.
2.5 statistical procedures
Data analysis is carried out using statistic software SPSS 21.0, generally uses variance analysis, but need to be by the journey of variance analysis
Sequence first carries out homogeneity test of variance, and variance is neat, calculates F values, F values<F0.05, conclusion：No significant difference between each group mean；F values
≥F0.05, P≤0.05 is counted with the comparative approach two-by-two of mean between multiple experimental groups and a control group；To abnormal
Or the data of heterogeneity of variance carry out variable conversion appropriate, after meeting normal state or variance requires together, with transformed data into
Row statistics；If being still not up to normal state or the neat purpose of variance after variable conversion, uses rank sum test instead and counted.
3 results
By table 5 as it can be seen that the left and right auricle weight difference of the mouse of each dosage group of Qi stilbene piece and levamisol group is apparently higher than
Physiological saline group, wherein low dose of group of Qi stilbene and levamisol group have significant difference (P ＜ 0.01), prompt Qi stilbene piece to mouse
Delayed allergy effect has apparent facilitation.
Influence of the 5 Qi stilbene piece of table to mouse delayed allergy (DTH)
Note：Compared with physiological saline group,*P ＜ 0.05,**P ＜ 0.01
4 brief summaries
Qi stilbene piece continuous gavage gives mouse 30 days, has apparent facilitation, and significant difference to mouse DTH.
Two, humoral immune function measures
(1) influence (Hemagglutination Method) of the Qi stilbene piece to mice serum hemolysin
1 test material
1.1 main agents
(Dongguan City Asia Pharmaceutical Co., Ltd provides Qi stilbene piece, lot number：20160614), Content of Tablet of Levamisole Hydrochloride (Guangdong
South China medicine company Group Co., Ltd, Chinese medicines quasi-word H44020759), 2% sheep red blood cell (SRBC) (the limited public affairs of Guangzhou letter spring biotechnology
Department, product batch number：160926), NaCl (Tianjin Zhi Yuan chemical reagent Co., Ltd, lot number：2016102067).
1.2 drug
Qi stilbene piece, 7.00g crude drugs/g, Qi stilbene piece clinic are 23.1g crude drugs/d (3.85g crude drugs/0.55g/ per consumption per day
Grain, one time 3, two times a day), for adult's weight in terms of 60kg, average dosage is 0.385g crude drugs/kg/d, this test mice
Basic, normal, high three dosage groups are set to 3.85,7.7,15.4g crude drugs/kg/d, above-mentioned dosage be clinical medicine dose 10,
20,40 times (according to the weight), levamisol：This experiment mice dose design is 25mg/kg.
1.3 animal
KM mouse, male, SPF grades, 18~22g, Nanfang Medical Univ's Experimental Animal Center, the animal quality quality certification：
SCXK (Guangdong) 2013-0002.
1.4 feeding environment：23.0 ± 2.0 DEG C, relative humidity 40-70% of experiment periods animal house environment temperature, rate of ventilation>
15 times/hour ,/12 hours dark of illumination in 12 hours, light and shade alternating, experimental animal use credit number：SYXK (Guangdong) 2012-
1125。
1.5 main equipment
Table-type low-speed large capacity centrifuge L550 (Hunan Xiang Yi Laboratory Instruments development corporation, Ltd.)
Microhemagglutination experimental plate (U.S. Corning).
Electro-heating standing-temperature cultivator (Shanghai Yiheng Scientific Instruments Co., Ltd)
2 methods
2.1 solution are prepared
0.5%SRBC：The 2%SRBC for taking 15mL, adds physiological saline to 60mL.
2.2 groupings and administration
Male KM mouse 60, weight 18-22g is taken, 5 groups, i.e. physiological saline group, left-handed miaow are randomly divided by weight
Basic, normal, high 3 dosage groups of azoles positive drug group, Qi stilbene piece (3.85,7.7,15.4g crude drugs/kg), every group 12.Qi stilbene piece is each
Every animal of dosage group and levamisol positive drug group gives the drug of corresponding dosage daily, and gavage volume is 20mL/kg, raw
Reason brine group then gives isometric physiological saline, successive administration 30d.
2.3 Hemagglutination Method
2.3.1 animal and serum separation is immunized
It is administered the 15th day, every mouse peritoneal injection 0.2mL 2%SRBC is immunized.After 5 days immune, after 1h is administered,
It weighs, angular vein clump takes blood in centrifuge tube, places about 1h, and solidification blood and tube wall are removed, serum is made fully to be precipitated,
2000r/min centrifuges 10min, collects serum.
2.3.2 agglutinating reaction
With physiological saline by serum doubling dilution (1:2~1:256) serum of different dilutions, is respectively placed in Trace Blood
In solidifying experimental plate, per 100 μ L of hole, the SRBC suspensions of 100 μ L 0.5% (v/v) are added, mixing is packed into the square position of moistening and adds
Lid observes hemagglutination degree in 37 DEG C of incubation 3h.
Serum agglutination degree is generally divided into 5 grades (0-IV) record, and antibody product is calculated as follows, tested Qi stilbene piece group
Antibody product is significantly higher than the antibody level of control group, can determine that this experimental result positive.
Antibody product=(S1+2S2+3S3……nSn)
1,2,3 ... n represent the index of two-fold dilution in formula, and the rank of S generation agglutination degree, antibody product is bigger, indicates
Serum antibody is higher.
0 grade of red blood cell all sinks, and concentrates on hole bottom and forms fine and close round point shape, surrounding liquid clear.
Largely for heavy collection in bottom hole at round spot shape, surrounding has the red blood cell being aggregated on a small quantity to I grade of red blood cell.
The red blood cell of II grade of agglutination forms thin layer in bottom hole, and center can obviously see a loose red point.
The uniform shakedown of red blood cell of III grade of agglutination is dispersed in bottom hole into a thin layer, and may be seen indistinctly a small red dot at center.
Uniformly paving is dispersed in bottom hole into a thin layer to the red blood cell of IV grade of agglutination, and grumeleuse is sometimes at convolution shape.
2.4 statistical procedures
Data analysis is carried out using statistic software SPSS 21.0, generally uses variance analysis, but need to be by the journey of variance analysis
Sequence first carries out homogeneity test of variance, and variance is neat, calculates F values, F values<F0.05, conclusion：No significant difference between each group mean；F values
≥F0.05, P≤0.05 is counted with the comparative approach two-by-two of mean between multiple experimental groups and a control group；To abnormal
Or the data of heterogeneity of variance carry out variable conversion appropriate, after meeting normal state or variance requires together, with transformed data into
Row statistics；If being still not up to normal state or the neat purpose of variance after variable conversion, uses rank sum test instead and counted.
3 results
By table 6 as it can be seen that the antibody product of each dosage group mouse of Qi stilbene piece is above physiological saline group, the wherein high agent of Qi stilbene piece
Amount group and levamisol group have significant difference (P ＜ 0.05 or P ＜ 0.01), and Qi stilbene piece is prompted to be known as mice serum haemolysis
Certain facilitation.
Influence of the 6 Qi stilbene piece of table to mice serum hemolysin moderate resistance volume number
Note：Compared with physiological saline group,*P ＜ 0.05,**P ＜ 0.01
4 brief summaries
Mouse continuously gives Qi stilbene piece gavage 30 days, there is apparent promotion to make the antibody product of mice serum blood lysin
With.The result shows that Qi stilbene piece has apparent facilitation to mice serum hemolytic antibody level.
Three, monokaryon-macrophage function measures
(1) influence of the Qi stilbene piece to mouse monokaryon-macrophage carbonic clearance function
1 test material
1.1 main agents
(Dongguan City Asia Pharmaceutical Co., Ltd provides Qi stilbene piece, lot number：20160614), Content of Tablet of Levamisole Hydrochloride (Guangdong
South China medicine company Group Co., Ltd, Chinese medicines quasi-word H44020759), india ink (Beijing Suo Laibao Science and Technology Ltd, production
Lot number：1224D023), natrium carbonicum calcinatum (Tianjin great Mao chemical reagent factories, product batch number：20151006).
1.2 drug
Qi stilbene piece, 7.00g crude drugs/g, Qi stilbene piece clinic are 23.1g crude drugs/d (3.85g crude drugs/0.55g/ per consumption per day
Grain, one time 3, two times a day), for adult's weight in terms of 60kg, average dosage is 0.385g crude drugs/kg/d, this test mice
Basic, normal, high three dosage groups are set to 3.85,7.7,15.4g crude drugs/kg/d, above-mentioned dosage be clinical medicine dose 10,
20,40 times (according to the weight), levamisol：This experiment mice dose design is 25mg/kg.
1.3 animal
KM mouse, male, SPF grades, 18~22g, Nanfang Medical Univ's Experimental Animal Center, the animal quality quality certification：
SCXK (Guangdong) 2013-0002.
1.4 feeding environment：23.0 ± 2.0 DEG C, relative humidity 40-70% of experiment periods animal house environment temperature, rate of ventilation>
15 times/hour ,/12 hours dark of illumination in 12 hours, light and shade alternating, experimental animal use credit number：SYXK (Guangdong) 2012-
1125.1.5 key instrument
Tecan Infinite 200PRO multi-function microplate readers (Tecan Austria GmbH).2 methods
2.1 solution are prepared
Injection ink：India ink stoste 8mL is taken, sets in the dry bottle of cleaning, physiological saline 20mL, i.e. India is added
3.5 times of ink dilution is spare.
0.1%Na2CO3Solution：Take 0.2g Na2CO3, add distilled water to 200mL.
2.2 groupings and administration
Male KM mouse 60, weight 18-22g is taken, 5 groups, i.e. physiological saline group, left-handed miaow are randomly divided by weight
Basic, normal, high 3 dosage groups of azoles positive drug group, Qi stilbene piece (3.85,7.7,15.4g crude drugs/kg), every group 12.Qi stilbene piece is each
Every animal of dosage group and levamisol positive drug group gives the drug of corresponding dosage daily, and gavage volume is 20mL/kg, raw
Reason brine group then gives isometric physiological saline, successive administration 30d.
2.3 carbonic clearances are tested
It after last dose 1h, weighs, injects diluted india ink from mouse tail vein and (use normal saline dilution 3.5
Times, now prepare before use), it is calculated on an empty stomach per 10g weight 0.1mL by mouse.Wait for that prepared Chinese ink injects, timing immediately.After injecting prepared Chinese ink
2,10min takes 20 μ L of blood from angular vein clump respectively, is added into 2mL 0.1%Na immediately2CO3In solution, mixing is blown and beaten.With
Microplate reader densitometric value (OD values) at 600nm wavelength, with Na2CO3Solution makees blank control.It is de- after blood sampling finishes twice
Cervical vertebra puts to death mouse, takes liver and spleen, blots organ surface blood stains with filter paper, weigh respectively.Mouse is indicated with phagocytic index
The ability of carbonic clearance, is calculated as follows phagocytic index (a).
Carbonic clearance index
Phagocytic index
2.4 statistical procedures
Data analysis is carried out using statistic software SPSS 21.0, generally uses variance analysis, but need to be by the journey of variance analysis
Sequence first carries out homogeneity test of variance, and variance is neat, calculates F values, F values<F0.05, conclusion：No significant difference between each group mean；F values
≥F0.05, P≤0.05 is counted with the comparative approach two-by-two of mean between multiple experimental groups and a control group；To abnormal
Or the data of heterogeneity of variance carry out variable conversion appropriate, after meeting normal state or variance requires together, with transformed data into
Row statistics；If being still not up to normal state or the neat purpose of variance after variable conversion, uses rank sum test instead and counted.
3 results
By table 7 as it can be seen that the middle and high dosage group of Qi stilbene piece and the phagocytic index of levamisol group mouse are apparently higher than physiological saline
Group has significant difference (P ＜ 0.05 or P ＜ 0.01), prompts Qi stilbene piece that can significantly improve the carbon of mouse monokaryon-macrophage
Clean up ability.
Influence of the 7 Qi stilbene piece of table to mouse monokaryon-macrophage carbonic clearance ability
Note：Compared with physiological saline group,*P ＜ 0.05,**P ＜ 0.01
4 brief summaries
Qi stilbene piece continuous gavage gives mouse 30 days, has to the carbonic clearance ability of mouse monokaryon-macrophage apparent
Effect.Show that Qi stilbene piece can enhance mouse monokaryon-macrophage phagocytosis Scavenging activity.
Four, NK cytoactive detections
(1) influence (LDH method) of the Qi stilbene piece to NK cell activity
1 test material
1.1 main agents
(Dongguan City Asia Pharmaceutical Co., Ltd provides Qi stilbene piece, lot number：20160614), YAC-1 cells are (by Guangdong medicine section
Basis institute of university provide), RPMI1640 culture solutions (Gibco, lot number：8116420), calf serum (Zhejiang day Hangzhoupro biology section
Skill Co., Ltd, lot number：20160721), 2 mercapto ethanol (Genview, lot number：6526010150), dual anti-(Solarbio,
Lot number：20160509), Hank's liquid (Genview, lot number：61121010110), PBS buffer solution (Hyclone, lot number：
AB10111039), trypan blue (the Guangzhou bio tech ltd Xiang Bo, lot number：0108XB15), hydrochloric acid (Tianjin great Mao reagents
Factory, lot number：20160715), lithium lactate (Sigma, lot number：MKBV2142V)；Nitro tetrazolium chloride (INT) (Sigma, batch
Number：SLBR2464V)；Phenazine Dimethyl Sulfate (PMS) (Sigma, lot number：P920271116)；Oxidized coenzyme I (NAD)
(Roche, lot number：L5300141), Tris-Hcl (Beyotime, lot number：091316161108), NP40 (Fluka, lot number：
SLNM1610E)、NH4Cl (Guangzhou Chemical Reagent Factory, lot number：20150504-2)、KHCO3(Guangzhou Chemical Reagent Factory, lot number：
2015Q401-1), EDTA (the good science and technology of prestige, lot number：20110718).
1.2 drug
Qi stilbene piece, 7.00g crude drugs/g, Qi stilbene piece clinic are 23.1g crude drugs/d (3.85g crude drugs/0.55g/ per consumption per day
Grain, one time 3, two times a day), for adult's weight in terms of 60kg, average dosage is 0.385g crude drugs/kg/d, this test mice
Basic, normal, high three dosage groups are set to 3.85,7.7,15.4g crude drugs/kg/d, above-mentioned dosage be clinical medicine dose 10,
20,40 times (according to the weight).
1.3 animal
KM mouse, SPF grades, 18~22g, Nanfang Medical Univ's Experimental Animal Center, the animal quality quality certification：SCXK (Guangdong)
2013-0002。
1.4 feeding environment：23.0 ± 2.0 DEG C, relative humidity 40-70% of experiment periods animal house environment temperature, rate of ventilation>
15 times/hour ,/12 hours dark of illumination in 12 hours, light and shade alternating, experimental animal use credit number：SYXK (Guangdong) 2012-
1125。
1.5 main equipment
HZT-A1000 electronic balances (Foochow Hua Zhi scientific instrument Co., Ltd), FA2004B electronic balances (upper Nereid section
Tian Mei scientific instrument Co., Ltd), Tecan Infinite 200PRO multi-function microplate readers (Tecan Austria GmbH),
Pulsation vacuum sterilizer (Xinhua Medical Apparatus Co., Ltd. Shandong), FA2104 upper ware electronic balances (Shanghai exact science
Instrument Ltd.), SW-CJ-1F clean benches (SuZhou Antai Air Tech Co., Ltd. of Su Jing groups), table-type low-speed it is big
Capacity centrifugation machine L550 (Hunan Xiang Yi Laboratory Instruments development corporation, Ltd.), blood counting chamber (Shanghai City refinement biochemical reagents
Instrument Ltd.), BA310-T biomicroscopes (Motic), AE2000 biomicroscopes (Motic), 3100 carbon dioxide training
Support case (Thermo), 96 orifice plate of the bottoms U (Corning), flat 96 orifice plate (Nest).
2 methods
2.1 solution are prepared：
The preparation of LDH matrix liquids：
Lithium lactate 5 × 10-2mol/L
Nitro tetrazolium chloride (INT) 6.6 × 10-4mol/L
Phenazine Dimethyl Sulfate (PMS) 2.8 × 10-4mol/L
Oxidized coenzyme I (NAD) 1.3 × 10-3mol/L
Mentioned reagent is dissolved in the Tris-HCl buffer solutions of 0.2mol/L (PH=8.2)
2.2 groupings and administration
Female KM mice 48, weight 18-22g is taken, 4 groups, i.e. physiological saline group, Qi stilbene piece are randomly divided by weight
Basic, normal, high 3 dosage groups (3.85,7.7,15.4g crude drugs/kg), every group 12.Qi stilbene piece every animal of each dosage group is daily
The drug of corresponding dosage is given, gavage volume is 20mL/kg, and physiological saline group then gives isometric physiological saline, successive administration
30d。
2.3 target cells pass on (YAC-1 cells)
Target cell is subjected to secondary culture for 24 hours before experiment.It is washed 3 times with Hank ' s liquid with preceding, is cultivated completely with RPMI1640
It is 1 × 10 that liquid, which adjusts cell concentration,5A/mL.
The preparation (effector cell) of 2.4 splenocyte suspensions
It is sterile at the end of experiment to take spleen, it is placed in the sterilized petri dishes for filling appropriate sterile Hank ' s liquid, with 4 layers of gauze by spleen
It grinds, individual cells suspension is made.It is filtered through 200 mesh screens, centrifugation 10min (1000r/min).Supernatant is abandoned, it is red thin that 1mL is added
Cellular lysate liquid blows and beats mixing, immediately timing, centrifugation 10min (1000r/min) after 5min.2 times are washed with Hank's liquid, every time again
10min (1000r/min) is centrifuged, supernatant is abandoned, is resuspended with RPMI1640 complete culture solutions of the 2mL containing 10% calf serum, uses platform
Expect blue dyeing counting viable count (should be 95% or more), finally use RPMI1640 complete culture solutions adjustment cell concentration be 5 ×
106A/mL.
2.5NK cytoactive detection
Target cell and each 100 μ L of effector cell are taken, is added in U-shaped 96 well culture plate, target cell Spontaneous release hole adds target thin
Born of the same parents and each 100 μ L of 1640 culture medium, target cell maximum release aperture add target cell and each 100 μ L of 1%NP40；Above-mentioned items are all provided with three
A parallel hole, in 37 DEG C, 5%CO24h is cultivated in incubator, 96 well culture plates is then centrifuged into 5min with 1500r/min, per hole
It draws in 96 well culture plate of supernatant 100uL horizontalizations bottoms, while LDH matrix liquid 100uL is added, according to room temperature differential responses 3min,
The HCl 30uL of 1mol/L are added per hole, the work of NK cells is calculated as follows in densitometric (OD) value at microplate reader 490nm wavelength
Property.
NK cell activity %=(reacting hole OD- Spontaneous releases hole OD)/(maximum release aperture OD- Spontaneous releases hole OD) ×
100%
2.6 statistical procedures
Data analysis is carried out using statistic software SPSS 21.0, generally uses variance analysis, but need to be by the journey of variance analysis
Sequence first carries out homogeneity test of variance, and variance is neat, calculates F values, F values<F0.05, conclusion：No significant difference between each group mean；F values
≥F0.05, P≤0.05 is counted with the comparative approach two-by-two of mean between multiple experimental groups and a control group；To abnormal
Or the data of heterogeneity of variance carry out variable conversion appropriate, after meeting normal state or variance requires together, with transformed data into
Row statistics；If being still not up to normal state or the neat purpose of variance after variable conversion, uses rank sum test instead and counted.
3 results：
By table 8 as it can be seen that the NK cell activity of each dosage group mouse of Qi stilbene piece is above physiological saline group, and in Qi stilbene piece,
High agent group has significant difference (P ＜ 0.05 or P ＜ 0.01), prompts Qi stilbene piece to have the NK cell killing activities of mouse bright
Aobvious facilitation.
Influence of the 8 Qi stilbene piece of table to NK cells in mice killing activity
Note：Compared with physiological saline group,*P ＜ 0.05,**P ＜ 0.01
4 brief summaries
Qi stilbene piece continuous gavage gives mouse 30 days, has apparent facilitation to the killing activity of NK cells in mice.
Five, it summarizes
One, cellular immune function assay
(1) influence of the Qi stilbene piece to mouse immune organ：Qi stilbene piece has apparent enhancing to the immune organ weight of mouse
Effect.(2) influence of the Qi stilbene piece to the mouse ConA Splenic vein hemodynamics induced：Qi stilbene piece drenches the spleen that mouse ConA is induced
Bar cell transformation has apparent facilitation.
(3) influence (ear swelling method) of the Qi stilbene piece to mouse delayed allergy (DTH)：Qi stilbene piece is to mouse delayed
Allergy effect has apparent facilitation.
As a result prompt Qi stilbene piece can enhance the cellular immune function of body.
Two, humoral immune function measures
Influence (Hemagglutination Method) of the Qi stilbene piece to mice serum hemolysin：Qi stilbene piece has mice serum hemolytic antibody level
There is apparent facilitation.As a result prompt Qi stilbene piece has the humoral immunity effect of certain enhancing body.
Three, monokaryon-macrophage function measures
Influence of the Qi stilbene piece to mouse monokaryon-macrophage carbonic clearance function：It is thin that Qi stilbene piece can enhance mouse monokaryon-macrophage
Endocytosis bites Scavenging activity.
Four, NK cytoactive detections
Influence (LDH method) of the Qi stilbene piece to NK cell activity：Qi stilbene piece has significantly the killing activity of NK cells in mice
Facilitation.
In conclusion this results show Qi stilbene piece has the effect that：
1, enhance the cellular immune function of body.
2, the humoral immunity effect of enhancing body.
3, enhance mouse monokaryon-macrophage and swallow Scavenging activity.
4, enhance NK cells in mice killing activity.
The composition of the present invention can be made ordinary preparation, can also be sustained release preparation, controlled release preparation, targeting preparation and each
Kind particulate delivery system.
In order to which tablet is made in unit dosage forms for administration, various carriers well known in the art can be widely used.About carrier
Example be, such as diluent and absorbent：Starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urine
Element, calcium carbonate, white bole, microcrystalline cellulose, alumina silicate etc.；Wetting agent and adhesive：Water, glycerine, polyethylene glycol, ethyl alcohol, third
Alcohol, starch slurry, dextrin, syrup, honey, glucose solution, mucialga of arabic gummy, gelatine size, sodium carboxymethylcellulose, lac, first
Base cellulose, potassium phosphate, polyvinylpyrrolidone etc.；Disintegrant, such as dry starch, alginate, agar powder, brown alga are formed sediment
Powder, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol aliphatic ester, dodecyl sodium sulfate, Methyl cellulose
Element, ethyl cellulose etc.；Disintegration inhibitor, such as sucrose, glyceryl tristearate, cocoa butter, ammonification oil；Sorbefacient,
Such as quaternary ammonium salt, lauryl sodium sulfate etc.；Lubricant, such as talcum powder, silica, cornstarch, stearate, boron
Acid, atoleine, polyethylene glycol etc.；Tablet can also further be made to coating tablet, for example, sugar coated tablet, thin membrane coated tablet,
Enteric coated tablets or double-layer tablets and multilayer tablet.
In order to which pill is made in administration unit, various carriers well known in the art can be widely used.Example about carrier
Be, such as diluent and absorbent, as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone,
Gelucire, kaolin, talcum powder etc.；Adhesive, as Arabic gum, bassora gum, gelatin, ethyl alcohol, honey, liquid sugar, rice paste or
Batter etc.；Disintegrant, such as agar powder, dry starch, alginate, dodecyl sodium sulfate, methylcellulose, ethyl cellulose
Deng.
In order to which capsule is made in administration unit, active ingredient is mixed with above-mentioned various carriers, and will be thus obtained
Mixture is placed in hard gelatine capsule or soft capsule, also can microcapsules be made in active ingredient, is suspended in shape in aqueous medium
At suspension, injection application also can be fitted into hard capsule or is made.
Such as injection preparation is made in the composition of the present invention, such as solution, suspension solution, emulsion, freeze-dried powder
Injection, this preparation can be aqueous or non-aqueous, can contain acceptable carrier in a kind of and/or a variety of pharmacodynamics, diluent,
Adhesive, lubricant, preservative, surfactant or dispersant, as diluent can be selected from water, ethyl alcohol, polyethylene glycol, 1,3- third
Glycol, the isooctadecanol of ethoxylation, polyoxygenated isooctadecanol, Polyoxyethylene Sorbitol Fatty Acid Esters etc..In addition, in order to make
Standby isotonic injection, can add suitable sodium chloride, glucose or glycerine, further, it is also possible to add into injection preparation
Conventional cosolvent, buffer, pH adjusting agent etc..These auxiliary materials are commonly used in the art.
In addition, if desired, can also be added into pharmaceutical preparation colorant, preservative, fragrance, corrigent, sweetener or
Other materials.
Preferably, the tablet the forming by following components parts by weight of the present invention：5~10 parts of the fruit of Chinese wolfberry, Radix Astragali 3~
8 parts, 1~6 part of ganoderma lucidum, 1~6 part of gynostemma pentaphylla, 4 parts of starch, 3 parts of honey, 2 parts of sodium bicarbonate, 1 part of sucrose, 1 part of talcum powder；On
Tablet is stated to be easy to suppress, meanwhile, the middle-aged and the old easily absorbs after taking, and effect is fine；It further, can also be further by tablet
Coating tablet, such as sugar coated tablet, thin membrane coated tablet, enteric coated tablets or double-layer tablets and multilayer tablet is made；Further, may be used also
Capsule is made, by 5~10 parts of the fruit of Chinese wolfberry, 3~8 parts of Radix Astragali, 1~6 part of ganoderma lucidum, 1~6 part of gynostemma pentaphylla, 4 parts of starch, honey 3
Part, 2 parts of sodium bicarbonate, 1 part of sucrose, 1 part of talcum powder, 1 part of methylcellulose are placed in hard gelatine capsule or soft capsule, also may be used
Microcapsules is made in active ingredient, is suspended in aqueous medium and forms suspension, also can be fitted into hard capsule or be made injection
Using.
Further, injection preparation can also be made in the health composition of the present invention, such as solution, suspension agent solution
Agent, emulsion, freeze drying powder injection, this preparation can be it is aqueous or non-aqueous, such as it is aqueous：5~10 parts of the fruit of Chinese wolfberry, Radix Astragali 3~8
Part, 1~6 part of ganoderma lucidum, 1~6 part of gynostemma pentaphylla, 4 parts of starch, 3 parts of honey, 3 parts of gelatin, 2 parts of sodium bicarbonate, 1 part of sucrose, talcum powder
1 part, 20-50 parts of water；Non-aqueous：5~10 parts of the fruit of Chinese wolfberry, 3~8 parts of Radix Astragali, 1~6 part of ganoderma lucidum, 1~6 part of gynostemma pentaphylla, starch 4
Part, 3 parts of honey, 2 parts of sodium bicarbonate, 1 part of sucrose, 1 part of talcum powder, 20-30 parts of 1,3-PD；In addition, isotonic in order to prepare
Injection can add suitable sodium chloride (0.5 part), glucose (1 part) or glycerine (1 part) into injection preparation, in addition,
Conventional cosolvent, buffer, pH adjusting agent etc. can also be added, these auxiliary materials are commonly used in the art.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than is protected to the present invention
The limitation of range is protected, although being explained in detail to the present invention with reference to preferred embodiment, those skilled in the art should
Understand, technical scheme of the present invention can be modified or replaced equivalently, without departing from the essence of technical solution of the present invention
And range.
Claims (5)
1. a kind of health composition, which is characterized in that the health composition is made of the raw material of following parts by weight：The fruit of Chinese wolfberry
5~10 parts；3~8 parts of Radix Astragali；1~6 part of ganoderma lucidum；1~6 part of gynostemma pentaphylla.
2. health composition as described in claim 1, which is characterized in that the health composition by following parts by weight original
Material is made：7 parts of the fruit of Chinese wolfberry, 6 parts of Radix Astragali, 5 parts of ganoderma lucidum, 5 parts of gynostemma pentaphylla.
3. the preparation method of health composition as described in claim 1, it is characterised in that：The preparation side of the health composition
Method includes the following steps：
A, raw material is weighed by following parts by weight：5~10 parts of the fruit of Chinese wolfberry；3~8 parts of Radix Astragali；1~6 part of ganoderma lucidum；Gynostemma pentaphylla 1~6
Part；
B, the fruit of Chinese wolfberry is taken to be extracted with water 1~4 time, 0.5~4 hour every time, filtration, merging filtrate concentrated, with 50~98wt%'s
Ethyl alcohol carries out alcohol precipitation separation；Taking precipitate is dried, crushed into fine powder；
C, Radix Astragali is taken to be extracted with water 1~4 time, 0.5~4 hour every time, filtration, merging filtrate concentrated, is dried, crushed into fine powder；
D, ganoderma lucidum is taken to be extracted 1~4 time with 30~98% alcohol reflux, 0.5~4 hour every time, filtration, merging filtrate recycled
Ethyl alcohol, concentration, obtains ethanol extract；The dregs of a decoction are extracted with water 1~4 time, and 0.5~4 hour every time, filtration, merging filtrate concentrated,
Obtain water extract；Two kinds of extracts are mixed, fine powder is dried, crushed into；
E, gynostemma pentaphylla is taken to be extracted with water 1~4 time, 0.5~4 hour every time, filtration, merging filtrate concentrated, with 50~98wt%'s
Ethyl alcohol carries out alcohol precipitation separation；It takes filtrate to concentrate, is dried, crushed into fine powder；
F, above-mentioned four kinds of fine powders are uniformly mixed and preparation is made.
4. application of the health composition according to claim 1 or 2 in preparing health products, it is characterised in that：The guarantor
Strong composition be used as give protection against cancer for strengthen immunity, anti-aging, auxiliary, answering in the health products of middle-aged and the old's daily health caring
With.
5. application of the health composition according to claim 4 in preparing health products, which is characterized in that the health products
For tablet, capsule, granule, pill, pastille, mixture, oral solution, powder, soft extract, syrup.
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