1, Lin Zhibin, the modern study of glossy ganoderma, publishing house of Beijing Medical University, March calendar year 2001.
2, State Administration of Traditional Chinese Medicine, " China's book on Chinese herbal medicine ", Shanghai science tech publishing house.
Summary of the invention
The purpose of this invention is to provide a kind of health food that strengthens immunity.
A further object of the invention provides the preparation technology of above-mentioned health food.
The objective of the invention is to realize by following technical measures:
A kind of health food that strengthens immunity, its prescription contains the raw material of following weight portion:
Propolis 50-60 part, Ganodenna Lucidum P.E 30-40 part, cordyceps mycelia 5-15 part.
Described health food, wherein Ganodenna Lucidum P.E prepares by following method: glossy ganoderma adds 8-12 times of water gaging after cutting into slices, extract 2-3 time, each 4-6 hour, merge extract, concentrate the back spray-drying; The quality requirement of Ganodenna Lucidum P.E is: polysaccharide 〉=8.0%, moisture≤8.0%, ash content≤10.0%.The outward appearance of Ganodenna Lucidum P.E is the dark-brown powder, drying, and free from admixture has the intrinsic fragrance of glossy ganoderma.
Health food with enhancing immunity of the present invention can add any one auxiliary material that pharmaceutically allows, its preparation can be any one formulation that pharmaceutically allows, include but not limited to tablet, granule, capsule, oral liquid, syrup, pill etc., can also add the health food that other materials that do not weaken health food effect of the present invention are made complex function in the health food of the present invention.
The preparation technology of described health food comprises the following step:
Get cordyceps mycelia by prescription and pulverize, cross 80 mesh sieves, mix with Ganodenna Lucidum P.E, 110-130 ℃ hot air sterilization 1-2 hour, add again through pulverizing the propolis of 80 mesh sieves, mix, common process is made required preparation.
Beneficial effect of the present invention:
Zoopery of the present invention:
One, material and method
1, sample: Chinese caterpillar fungus honeybee curing capsule, the 0.25g/ grain, the human body recommended intake is content net weight 1000mg/ people/day, and lot number 20040518 is provided by Nanjing Zhongke Biochemical Technology Co., Ltd.
2, animal used as test:
The I group: 40 of 18.9-23.7g ICR healthy male mices, be divided into 4 groups at random, 10 every group, carry out dinitrofluorobenzene inducing mouse DTH test;
The II group: 40 of 18.5-23.4g healthy male mices, be divided into 4 groups at random, 10 every group, carry out Turnover of Mouse Peritoneal Macrophages and engulf the chicken red blood cell test;
The III group: 40 of 18.3-23.5g healthy male mices, be divided into 4 groups at random, 10 every group, carry out the clearance test of mouse carbon;
The IV group: 40 of 20.0-23.9g healthy male mices, be divided into 4 groups at random, 10 every group, carry out mouse antibodies cellulation and HD50 value (HC
50) test;
The V group: 40 of 20.3-23.9g BALB/C healthy male mices, be divided into 4 groups at random, 10 every group, carry out mouse lymphocyte conversion, the test of NK cytoactive that mouse ConA induces.
3, dosage:
According to everyone (by the 60kg batheroom scale) recommended intake every day is 1000mg, be equivalent to 16.7mg/d/kgbw, press respectively 5 times of human body recommended intakes, 10 times, 30 times designs low (84mg/kgbw), in (167mg/kgbw), high (500mg/kgbw) three dosage groups, other establishes control group (0mg/kgbw) and replaces being tried thing with sterilized water.Per os gives the thing that tried of mouse corresponding dosage once a day, and the continuous irrigation stomach is surveyed every enhancing immunity function index after 30 days.The mouse stomach amount is 10ml/kgbw.
4, main agents: dinitrofluorobenzene (DNFB), sheep red blood cell (SRBC) (SRBC), GPS, india ink, RPMI1640, ConA, MTT, isopropyl alcohol, SA buffer solution, Dou Shi reagent, YAC-1 cell, LDH matrix liquid.
5, key instrument: card punch, T1000 type electric animal balance, JA2003 type electronic balance, 722 type spectrophotometers, superclean bench, ELIASA, CO2gas incubator.
6, test method: the enhancing immunity function method of inspection by " health food check with assessment technique standard-2003 edition " is carried out.
6.1ConA inducing mouse SPL conversion test (mtt assay):
Each treated animal continuous irrigation stomach 30 days, animal is put to death in the cervical vertebra dislocation, and the aseptic spleen of getting places the little plate that fills an amount of aseptic Hank ' s liquid, grind spleen, make the individual cells suspension, filter, use Hank ' s liquid to wash 3 times through 200 eye mesh screens, each centrifugal 10min (1000r/min), then cell is suspended in the 1ml RPMI1640 complete culture solution, the blue dyeing counting viable count of platform phenol (should more than 95%), adjusting cell concentration is 3 * 10
6Individual/ml.Divide two holes to add in 24 culture plates cell suspension, every hole 1ml, a hole adds 75 μ l ConA liquid, and another hole compares, and puts 5%CO
2Cultivate 72h in 37 ℃ of incubators.Cultivate and finish preceding 4h, supernatant 0.7ml is inhaled in every hole gently, adds the RPMI1640 nutrient solution that 0.7ml does not contain calf serum, adds MTT (5mg/ml) 50 μ l/ holes simultaneously, continues to cultivate 4h.After cultivating end, every hole adds 1ml acid isopropyl alcohol, and the piping and druming mixing dissolves purple crystal fully.Lysate is moved in 96 well culture plates, wavelength 570nm, ELIASA is measured the optical density evaluation.
6.2 the tardy parasexuality reaction of dinitrofluorobenzene (DNFB) inducing mouse (DTH) (ear swelling method):
Each dosage group continuous irrigation stomach 30 days, every mouse cuts off belly wool with scissors, the about 3cm * 3cm of scope, 50 μ l evenly smear sensitization with 1%DNFB solution.Evenly be applied in mouse right ear (two sides) with 1%DNFB solution 10 μ l after 5 days and attack, evenly be applied in mouse left side ear (two sides) with the solution 10 μ l that do not contain DNFB simultaneously and compare.Attack back 24h cervical vertebra dislocation and put to death mouse, cut left and right sides auricular concha, take off diameter 8mm auricle, weigh with card punch.Get mouse spleen, thymus gland simultaneously and weigh, calculate dirty/body ratio.
6.3 serum hemolysin is measured:
Each dosage group continuous irrigation stomach 30 days, sheep red blood cell (SRBC) (SRBC) suspension of preparation 2% (v/v), every mouse lumbar injection 0.2ml carries out immunity, extracts eyeball after 4 days and gets blood in centrifuge tube, places 1h, and the centrifugal 10min of 2000r/min separates and collection serum.After 200 times of dilutions of serum, the OD value during by method of inspection working sample pipe and SRBC HD50.The amount of hemolysin is with HD50 value (HC
50) expression.
6.4 antibody-producting cell detects (Jerne improves slide method):
Each treated animal continuous irrigation stomach 30 days, sheep red blood cell (SRBC) (SRBC) the suspension 0.2ml of every mouse lumbar injection 2% (v/v) carries out immunity, and the dislocation of mouse cervical vertebra is put to death after 4 days, take out spleen, be placed in the little plate that fills an amount of aseptic Hank ' s liquid, grind spleen, make cell suspension, filter through 200 eye mesh screens, centrifugal (1000r/min) 10min uses Hank ' s liquid to wash 2 times, at last cell is suspended in the 5ml RPMI1640 nutrient solution, the counting cells number, adjusting cell concentration is 5 * 10
6Individual/ml.After the culture medium heating for dissolving of top layer, put 45 ℃ of water bath heat preservations, mix with Hank ' the s liquid of equivalent pH7.2-7.4,2 times of concentration, the packing small test tube, every pipe 0.5ml adds 50 μ l, 10% (v/v again in pipe, with the preparation of SA liquid) SRBC, 20 μ l splenocyte suspensions (5 * 10
6Individual/ml), rapidly mixing is poured on the slide of brushing the agarose thin layer, treat that agar solidifies after, the slide level buckled be placed on the horse, put into CO
2Hatch 1.5h in the incubator, will join in the slide frame groove after the complement dilution in 1: 10 that prepare then, after continuing to hatch 1.5h, counting hemolysis plaque number.
6.5 mouse carbon clearance test:
Each dosage group continuous irrigation stomach 30 days, every caudal vein injects the india ink (0.1ml/10gbw) of 3 times of dilutions, treats that prepared Chinese ink injects timing immediately.2min and 10min get blood 20 μ l from intraocular corner of the eyes veniplex respectively behind the injection prepared Chinese ink, and it is added to 2ml 0.1%Na
2CO
3In the solution, with 722 spectrophotometers at 600nm wavelength place's photometry density value.
6.6 Turnover of Mouse Peritoneal Macrophages is engulfed chicken red blood cell test (half intracorporal method):
Each treated animal continuous irrigation stomach 30 days, the chicken erythrocyte suspension of preparation 20% (v/v), every mouse lumbar injection 1ml, interval 30min, mouse is put to death in the cervical vertebra dislocation, it is faced upward the position is fixed on the mouse plate, abdominal skin is cut off in the center, injects physiological saline 2ml through the abdominal cavity, rotates mouse plate 1min, sucking-off abdominal cavity washing lotion 1ml then, average mark drips on 2 slides, puts into the enamel box that is lined with wet gauze, 37 ℃ of incubation 30min of dislocation, rinsing in physiological saline then, dry, fix, 4% (v/v) Giemsa-phosphate buffer dyeing 3min with 1: 1 acetone methanol solution, dry with the distilled water rinsing, light microscopic is observed down again.
6.7NK cytoactive is measured (determination of lactate dehydrogenase method):
Each treated animal continuous irrigation stomach 30 days, mouse is put to death in the cervical vertebra dislocation, the aseptic spleen of getting, place the little plate that fills an amount of aseptic Hank ' s liquid, grind spleen, make single cell suspension, filter through 200 eye mesh screens, use Hank ' s liquid to wash 2 times, centrifugal at every turn (1000r/min) 10min abandons the supernatant friendship cytoplasm is upspring, and adds 0.5ml sterilization washing 20 seconds, add 0.5ml 2 times of Hank ' s liquid and 8ml Hank ' s liquid after the splitting erythrocyte again, centrifugal (1000r/min) 10min, the RPMI1640 complete culture solution that contains 10% calf serum with 1ml is resuspended, with 1% glacial acetic acid dilution back counting, the blue dyeing counting viable count of platform phenol (should more than 95%) is 2 * 10 with RPMI1640 complete culture solution adjustment cell concentration
7Individual/ml.
24h washes target cell (YAC-1 cell) cultivations of going down to posterity 3 times with Hank ' s liquid before using before the test, is 4 * 10 with RPMI1640 complete culture solution adjustment cell concentration
5Individual/ml.Getting each 100 μ l of YAC-1 cell and splenocyte (imitating target than 50: 1) adds in U type 96 well culture plates, YAC-1 cell nature release aperture adds YAC-1 cell and each 100 μ l of nutrient solution, the maximum release aperture of YAC-1 cell adds YAC-1 cell and each 100 μ l of 1%NP40, above-mentioned every three parallel holes of all establishing are in 37 ℃, 5%CO
2Cultivate 4h in the incubator, then with 96 well culture plates with the centrifugal 5min of 1500r/min, draw at the bottom of the supernatant 100 μ l horizontalizations in 96 well culture plates in every hole, add LDH matrix liquid 100 μ l simultaneously, reaction 5min, every hole adds the HCl 30 μ l of 1mol/L, measures OD value at ELIASA 490nm place.
7, raising condition: mouse is that 18-22 ℃, relative humidity are to raise in the barrier system of 40-70% in temperature.
8, data analysis: with SPSS10.0 software each experiment initial data is carried out homogeneity test of variance, satisfy the neat data information that requires of variance and carry out statistical disposition with the comparative approach in twos of mean between one-way analysis of variance method and a plurality of experimental group and control group; The data information of Non-Gaussian Distribution or heterogeneity of variance is carried out suitable variable conversion, wait to satisfy the normal state variance is neat require after, carry out statistical disposition with the data of changing gained.
Two, result:
1, Chinese caterpillar fungus honeybee curing capsule is to the influence of mouse body weight
By table 1 as seen, the initial body weight of mouse is compared with control group in basic, normal, high three dosage groups, and there are no significant for difference.The initial body weight of mouse is comparatively balanced between each group.Per os gives the Chinese caterpillar fungus honeybee curing capsule 30 days of mouse various dose, each dosage group body weight is carried out homogeneity test of variance, satisfy the requirement of homogeneity of variance, carried out statistical disposition with the comparative approach in twos of mean between one-way analysis of variance method and a plurality of experimental group and control group.Compared with control group by visible basic, normal, high three the dosage groups of table 2 result, there are no significant for difference.Be that Chinese caterpillar fungus honeybee curing capsule does not have influence to weight of mice.
Table 1 is respectively organized initial body weight (n=10, the x ± SD) of mouse
| Immunity I group | Immunity II group | Immunity III group | Immunity IV group | Immunity V group |
Control group | 20.9±1.6 | 20.9±1.6 | 21.0±1.7 | 22.1±1.1 | 22.1±1.0 |
Low dose group | 20.9±1.6 | 20.9±1.6 | 21.0±1.6 | 22.1±1.1 | 22.1±1.0 |
Middle dosage group | 20.9±1.6 | 20.9±1.6 | 20.9±1.6 | 22.1±1.1 | 22.1±1.0 |
High dose group | 20.9±1.7 | 20.9±1.6 | 20.9±1.6 | 22.1±1.2 | 22.1±1.1 |
Annotate: each is organized the P value and is 1.000.
Table 2 Chinese caterpillar fungus honeybee curing capsule is to the influence of mouse body weight (n=10, x ± SD)
| Immunity I group | Immunity II group | Immunity III group | Immunity IV group | Immunity V group |
Control group | 28.8±4.7 | 28.5±3.7 | 30.1±5.5 | 29.4±2.5 | 29.5±2.0 |
Low dose group | 29.0±3.7 | 30.1±3.8 | 29.9±4.7 | 30.6±1.7 | 30.1±1.1 |
Middle dosage group | 28.2±4.2 | 29.3±4.9 | 31.5±4.1 | 29.7±1.7 | 29.2±2.9 |
High dose group | 26.3±4.2 | 31.8±4.4 | 29.8±5.8 | 29.6±2.7 | 30.2±1.8 |
Annotate: each organizes P>0.05.
2, Chinese caterpillar fungus honeybee curing capsule is to the influence of thymus gland, spleen organ
Per os gives the Chinese caterpillar fungus honeybee curing capsule 30 days of mouse various dose, the chest of being measured/body ratio, spleen/body ratio carry out normal distribution, homogeneity test of variance, satisfy the requirement of normal distribution, homogeneity of variance, carried out statistical disposition with the comparative approach in twos of mean between one-way analysis of variance method and a plurality of experimental group and control group.Compared with control group by visible basic, normal, high three the dosage groups of table 3 result, there are no significant for difference.
Table 3 Chinese caterpillar fungus honeybee curing capsule is to the influence of thymus gland, spleen organ (n=10, x ± SD)
Group | Thymus gland/body weight (mg/g) | P | Spleen/body weight (mg/g) | P |
Control group | 141±0.69 | | 5.43±1.49 | |
Low dose group | 1.00±0.23 | 0.099 | 6.06±1.38 | 0.674 |
Middle dosage group | 1.11±0.37 | 0.300 | 6.45±1.68 | 0.316 |
High dose group | 1.03±0.28 | 0.143 | 6.00±1.46 | 0.736 |
3, the influence that transforms of Chinese caterpillar fungus honeybee curing capsule mouse spleen lymphocyte that ConA is induced
Per os gives the Chinese caterpillar fungus honeybee curing capsule 30 days of mouse various dose, institute's measured value is carried out homogeneity test of variance, satisfy the homogeneity of variance requirement, carried out statistical disposition with the comparative approach in twos of mean between one-way analysis of variance method and a plurality of experimental group and control group.Add the ConA hole by visible basic, normal, high three the dosage group mouse of table 4 result and be significantly higher than control group with the difference that does not add ConA hole absorbance.
The influence that the mouse spleen lymphocyte that table 4 Chinese caterpillar fungus honeybee curing capsule is induced ConA transforms (n=10, x ± SD)
Group | Add ConA hole and the difference that does not add ConA hole absorbance | P |
Control group | 0.029±0.016 | |
Low dose group | 0.062±0.035 | 0.015 |
Middle dosage group | 0.072±0.025 | 0.001 |
High dose group | 0.068±0018 | 0.004 |
4, Chinese caterpillar fungus honeybee curing capsule is to the influence of DNFB inducing mouse DTH
Per os gives the Chinese caterpillar fungus honeybee curing capsule 30 days of mouse various dose, institute's measured value is carried out homogeneity test of variance, satisfy the homogeneity of variance requirement, carried out statistical disposition with the comparative approach in twos of mean between one-way analysis of variance method and a plurality of experimental group and control group.Be significantly higher than control group by the weightening finish of the visible high dose group mouse of table 5 result auricular concha.
Table 5 Chinese caterpillar fungus honeybee curing capsule is to the influence of DNFB inducing mouse DTH (n=10, x ± SD)
Group | The auricular concha weightening finish | P |
Control group | 5.0±3.2 | |
Low dose group | 5.8±2.2 | 0.871 |
Middle dosage group | 7.6±2.9 | 0.129 |
High dose group | 9.6±3.2 | 0.003 |
5, Chinese caterpillar fungus honeybee curing capsule is to the influence of mice serum hemolysin formation
Per os gives the Chinese caterpillar fungus honeybee curing capsule 30 days of mouse various dose, institute's measured value is carried out homogeneity test of variance, satisfy the homogeneity of variance requirement, carried out statistical disposition with the comparative approach in twos of mean between one-way analysis of variance method and a plurality of experimental group and control group.Be significantly higher than control group by the visible high dose group mice serum of table 6 result hemolysin content.
The influence that table 6 Chinese caterpillar fungus honeybee curing capsule forms the mice serum hemolysin (n=10, x ± SD)
Group | HC
50 | P |
Control group | 56±43 | |
Low dose group | 107±54 | 0.082 |
Middle dosage group | 108±57 | 0.076 |
High dose group | 123±50 | 0.017 |
6, the influence of Chinese caterpillar fungus honeybee curing capsule antagonist cellulation (hemolysis plaque number)
Per os gives the Chinese caterpillar fungus honeybee curing capsule 30 days of mouse various dose, institute's measured value is carried out normal distribution, homogeneity test of variance, satisfy the normal distribution requirement, carried out statistical disposition with the comparative approach in twos of mean between one-way analysis of variance method and a plurality of experimental group and control group.Be higher than control group by visible middle and high two the dosage group mouse hemolysis plaque digital display works of table 7 result.
Table 7 Chinese caterpillar fungus honeybee curing capsule is to the influence of mouse antibodies cellulation (hemolysis plaque number) (n=10, x ± SD)
Group | Hemolysis plaque number (individual/10
6Splenocyte)
| P |
Control group | 119±15 | |
Low dose group | 163±53 | 0.217 |
Middle dosage group | 204±61 | 0.006 |
High dose group | 208±80 | 0.004 |
7, Chinese caterpillar fungus honeybee curing capsule is cleaned up the influence of effect to mouse carbon
Per os gives the Chinese caterpillar fungus honeybee curing capsule 30 days of mouse various dose, institute's measured value is carried out homogeneity test of variance, satisfy the homogeneity of variance requirement, carried out statistical disposition with the comparative approach in twos of mean between one-way analysis of variance method and a plurality of experimental group and control group.Compared with control group by visible basic, normal, high three the dosage groups of table 8 result, there are no significant for difference.
Table 8 Chinese caterpillar fungus honeybee curing capsule is cleaned up influence (n=10, the x ± SD) of effect to mouse carbon
Group | Phagocytic index a | P |
Control group | 4.48±1.66 | |
Low dose group | 4.80±1.22 | 0.893 |
Middle dosage group | 4.46±1.26 | 1.000 |
High dose group | 4.42±0.64 | 0.999 |
8, Chinese caterpillar fungus honeybee curing capsule is engulfed the phagocytic rate of chicken red blood cell and the influence of phagocytic index to Turnover of Mouse Peritoneal Macrophages
Per os gives the Chinese caterpillar fungus honeybee curing capsule 30 days of mouse various dose, the phagocytic rate of measuring, phagocytic index are carried out homogeneity test of variance, satisfy the homogeneity of variance requirement, carried out statistical disposition with the comparative approach in twos of mean between one-way analysis of variance method and a plurality of experimental group and control group.Compared with control group by visible basic, normal, high three the dosage groups of table 9 result, there are no significant for difference.
Table 9 Chinese caterpillar fungus honeybee curing capsule is engulfed the phagocytic rate of chicken red blood cell and the influence of phagocytic index (n=10, x ± SD) to Turnover of Mouse Peritoneal Macrophages
Group | Phagocytic rate (%) | P | Phagocytic index | P |
Control group | 20.3±4.7 | | 0.24±0.07 | |
Low dose group | 23.3±9.2 | 0.617 | 0.30±0.11 | 0.322 |
Middle dosage group | 22.9±5.7 | 0.710 | 0.28±0.08 | 0.587 |
High dose group | 24.1±5.9 | 0.438 | 0.31±0.06 | 0.174 |
9, Chinese caterpillar fungus honeybee curing capsule is to the influence of NK cells in mice activity
Per os gives the Chinese caterpillar fungus honeybee curing capsule 30 days of mouse various dose, institute's measured value is carried out homogeneity test of variance, satisfy the homogeneity of variance requirement, carried out statistical disposition with the comparative approach in twos of mean between one-way analysis of variance method and a plurality of experimental group and control group.Be significantly higher than control group by the visible high dose group NK cells in mice of table 10 result activity.
Table 10 Chinese caterpillar fungus honeybee curing capsule is to the influence of NK cells in mice activity (n=10, x ± SD)
Group | NK cytoactive (%) | P |
Control group | 31.1±8.2 | |
Low dose group | 35.1±6.8 | 0.496 |
Middle dosage group | 26.1±6.9 | 0.296 |
High dose group | 42.1±7.4 | 0.006 |