CN1251697C - Composition with functions of regulating immunity and resisting fatigue and its prepn, use and quality control method - Google Patents
Composition with functions of regulating immunity and resisting fatigue and its prepn, use and quality control method Download PDFInfo
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- CN1251697C CN1251697C CNB031192211A CN03119221A CN1251697C CN 1251697 C CN1251697 C CN 1251697C CN B031192211 A CNB031192211 A CN B031192211A CN 03119221 A CN03119221 A CN 03119221A CN 1251697 C CN1251697 C CN 1251697C
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Landscapes
- Medicines Containing Plant Substances (AREA)
Abstract
The present invention relates to a soft capsule prepared from extract of American ginseng saponin and alcohol extract of purple daisy, which can simultaneously perform the functions of immunological regulation and fatigue resistance. By research, the present invention also discloses a quality control method of the product further.
Description
Technical field
The present invention relates to a kind of compositions with immunomodulating and anti-fatigue effect, and preparation method thereof, purposes and method of quality control.
Background technology
General human body is fallen ill under two states easily, and the one, human body environment-adapting ability to external world descends, and body is in fatigue and weak state, and the one, be subjected to the infringement of external environment such as pathogen, immune function descends.And traditional prophylactic method is just taken the nourishing medicine merely to improve body condition, strengthens the adaptive capacity to environment, or improves the immunocompetence of body, to improve the human body resistance against diseases.
Radix Panacis Quinquefolii is the dry root of Araliaceae Radix Panacis Quinquefolii Panax quinquefolium L., be described as the representative that " strengthening the body resistance " reaches " adapting to the former state medicine ", can play the strengthening by means of tonics effect to general body, strengthen resistance against diseases, when physiological function disorder and imbalance appearred in sickened body, it has adjusted and therapeutical effect.Radix Panacis Quinquefolii China regarded as from ancient times the health care top grade, modern medicine has obvious anti-fatigue and immunological enhancement to the Radix Panacis Quinquefolii that studies show that of Radix Panacis Quinquefolii.
SONGGUOJU is the dry herb of the pale reddish brown SONGGUOJU Echinacea purpurea of feverfew, has another name called Echinacea, is America and Europe one of the most salable medicinal plants in recent years, has tangible immunological enhancement and immunoregulation effect.The America and Europe, in history with Echinacea as blood clean agent and immunostimulant and extensive use, and be used for the treatment of snake venom, hypoimmunity, leukopenia and all kinds of inflammation, be used for preventing cold and influenza more.Modern medicine study shows, Echinacea can produce many nonspecific effects to immune system, but as the activating complement alternative route, improve leukocytic quantity and strengthen leukocytic activity, promote the activation of non-specific T-cell, strengthen the phagocytic activity of macrophage etc., thereby the effect with tangible human body immunity improving function.Often take Echinacea with prevent disease and health invigorating American-European people.
But Echinacea does not have resisting fatigue and adapts to the former state effect, so it can only play auxiliary and unilateral effect to the people who has a delicate constitution.Just can improve the human body stress ability of environmental change to external world effectively and add Radix Panacis Quinquefolii, enhancing is to the adaptive capacity of environment, eliminate the weak state of body effectively and improve the human quality, so two kind of plant are used, coordinative role can be arranged on function, and better bring into play immunomodulating and anti-fatigue effect.
Summary of the invention
The application is exactly with Radix Panacis Quinquefolii and Echinacea compatibility, and a kind of compositions that can bring into play immunomodulating and anti-fatigue effect simultaneously is provided.By research, also further formulated the method for quality control of this product.
The raw material of product is formed and preparation technology: total panaquilon class crude extract 1-2 part, Echinacea 40-70% ethanol extraction 3-5 part, the optimum ratio of products material is: 1 part of total panaquilon class crude extract, 3.5 parts of Echinacea 40-70% ethanol extractions preferably use Echinacea 50% ethanol extraction.
This product can be made any dosage form of pharmaceutics, and as tablet, powder, granule, capsule, oral liquid, preferred dosage form is a soft capsule.
For soft capsule, its supplementary material prescription is as follows:
Total panaquilon class crude extract 1-2 part
Echinacea 40-70% ethanol extraction 3-5 part,
PEG400 5-10 part
Glycerol 1-3 part
Soybean salad oil 1-3 part
Water 1-3 part
Wherein PEG400, glycerol, soybean salad oil can replace with other corresponding pharmaceutics acceptable auxiliary.
The method of quality control of product and raw material
This method is applicable to Radix Panacis Quinquefolii, Echinacea or total panaquilon class extract, Echinacea ethanol extraction to be the quality control of the compositions made of raw material.
One, the total Saponin of functional component, Ginsenoside Rb in the compositions
1Assay
1. ginsenoside's content in the spectrophotometry compositions
Contained ginsenoside in the Radix Panacis Quinquefolii through the little column purification of macroporous adsorbent resin pretreatment, reacts with vanillin under acid condition, carries out quantitatively with spectrophotography.
The formulation of standard curve: get ginsenoside Re's standard substance, be mixed with the standard solution that concentration is 1g/L, get the standard solution different volumes, be settled to 1.0ml with 70% ethanol respectively, the accurate vanillin alcoholic solution 0.5ml that adds, 80% sulfuric acid solution 5ml shakes up, and puts in 60 ℃ ± 1 ℃ water-bath heating and develops the color to color when relatively stable, taking-up is put into ice bath immediately and is cooled off, colorimetric is measured trap in the 544nm place, with trap to concentration return regression equation.
The mensuration of sample: it is an amount of to get compositions, and precision takes by weighing, and extracts with suitable quantity of water, and it is standby to be settled to 100ml again, get this sample solution 0.5-1ml, cross macroporous resin column (this post has been used acetone, and water wash purifies), with 70% ethanol elution Radix Ginseng total saponins, collect eluent, stand-by.
With the accurate vanillin alcoholic solution 0.5ml that adds of above-mentioned eluent, 80% sulfuric acid solution 5ml, shake up, put in 60 ℃ ± 1 ℃ water-bath and heat, take out behind the color stability and put into ice bath immediately and cool off, colorimetric is measured trap in the 544nm place, in the content of ginsenoside of regression equation calculation with the ginsenoside Re.
2. high-efficient liquid phase technique is measured the content of the ginsenoside Rb1 in the product
Adopt the high performance liquid chromatography can be with the ginsenoside Rb1 with other component separating and quantitative.The ginsenoside Rb1 has absworption peak at the 203nm place, can carry out quantitative assay to Rb1.
Chromatographic condition: chromatographic column: ODS post (5m, 4.6 * 250nm); Mobile phase: acetonitrile-0.05% phosphoric acid solution (99: 400), flow velocity: 1ml/min detects wavelength: 203nm.
The mensuration of standard curve: precision takes by weighing ginsenoside Rb1's reference substance 10.0mg, with mobile phase dissolving and be settled in the 10ml volumetric flask, is reference substance solution.Draw reference substance solution different volumes sample introduction respectively, measure the data obtain peak area, with peak area to concentration return regression equation.
The mensuration of sample: it is an amount of that precision takes by weighing compositions, uses water dissolution, and water intaking solution filters, filtrate is an amount of, uses water saturated n-butanol extraction, merges the n-butyl alcohol phase, decompression and solvent recovery is to doing, and the residue dissolve with methanol filters, filtrate is used methanol constant volume, cross the microporous filter membrane of 0.35-0.50 μ m, the accurate absorption in right amount injected chromatograph of liquid, in the data of 203nm mensuration peak area, by the content of ginsenoside Rb1 in the regression equation calculation sample.
Two, to the qualitative identification of Echinacea in the compositions
Chemical constituent and pharmacological research to Echinacea show, Echinacea contains compositions such as polysaccharide, caffeic acid derivant, volatile oil, poly acetylene, alkylamide, but the health-care effect of any single component or component is all not obvious, and the synergism of each component is very strong, so the functional component of Echinacea also can't be determined at present.Some company that produces the Echinacea preparation is the index components of chicoric acid as Echinacea abroad, but chicoric acid is very unstable, does not have commercially available standard substance at present again, therefore can't carry out quantitative assay to Echinacea now.But we have carried out qualitative identification to Echinacea, contain the quality of Echinacea extract product with monitoring.
Contain polysaccharide composition inulin in the Echinacea, inulin is one of characteristic chemical constituent of feverfew, and this composition and α-naphthols, strong sulfuric acid response can generate the purple chemical compound and develop the color, in order to the check feverfew.
Contain multiple caffeoyl analog derivative in the Echinacea, wherein caffeic acid, chlorogenic acid are that its one-tenth is grouped into.In natural plants, most plants or only contain caffeic acid, or only contain chlorogenic acid, and the plant that contains these two kinds of compositions simultaneously yet there are no report, thus can carry out qualitative identification with these two kinds of compositions to Echinacea simultaneously, with outstanding its characteristic.Caffeic acid in the Echinacea, chlorogenic acid after thin layer chromatography separates, with the iodine colour developing, on the position corresponding with reference substance, show the speckle of same color.
Use these two kinds of methods to carry out qualitative identification to Echinacea extract.
1. the color reaction of Echinacea: it is an amount of to get compositions, with water extraction, draws aqueous solution and places test tube in right amount.Add 3 of α-naphthols alcoholic solution, shake up the tailing edge tube wall and slowly add concentrated sulphuric acid 1ml, between sulfuric acid layer and test liquid layer, occur tangible purplish red colour circle at the interface.
2. the thin layer chromatography of Echinacea: it is an amount of to get compositions, with water extraction, water intaking solution is an amount of, filters, with chloroform extraction, discard chloroform solution, water layer transfers to pH3.0 with dilute hydrochloric acid solution, the reuse ethyl acetate extraction, the combined ethyl acetate extract, the reclaim under reduced pressure ethyl acetate is to doing, and residue dissolves in right amount with methanol, is need testing solution.Preparation caffeic acid, chlorogenic acid concentration are the methanol solution product solution in contrast of 1.0-2.0mg/ml.Each is an amount of to get need testing solution and reference substance solution, point is on the thin layer of silica gel G, lower floor's solution with ethyl acetate-formic acid-water (10: 1: 9) launches, take out, fling to developing solvent, develop the color with iodine, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color, be caffeic acid and the chlorogenic acid that detects in the sample.
1,2 two kinds of qualitative identification methods are suitable for the alcohol extract of Echinacea is carried out qualitative identification equally.
Three, the quality standard of Echinacea raw material
For controlling product quality better, the reply raw materials quality is controlled.Quality control to the raw material Radix Panacis Quinquefolii adopts existent method and standard to get final product, and can adopt following qualitative method to control to the raw material Echinacea.
1, it is an amount of to get the Echinacea powder, places a few hours after adding the jolting of 60-75% ethanol, filters, and gets filtrate 2ml, adds 1～2 of 1% test solution, promptly produces dark blue precipitate.
2, get above-mentioned filtrate 1ml, place in vitro, add 3 of α-tea phenol alcoholic solution, shake up the tailing edge tube wall and slowly add concentrated sulphuric acid 1ml and between sulfuric acid layer and test liquid layer, occur tangible purplish red colour circle at the interface.
3, it is an amount of to get the Echinacea powder, and extracting in water filters, get filtrate 5ml, add ethanol and make the alcohol amount of containing reach 70-80%, jolting, leave standstill, filter, with an amount of washing precipitation of 70-80% ethanol, get the acclimatization thing, use water dissolution, it is a small amount of to add hydrochloric acid, the boiling water bath heating was transferred pH＞7 with sodium hydroxide test solution after 20-30 minute, it is a small amount of to add Fehling solution, and heating is several minutes on the boiling water bath, promptly produces brick-red precipitation.
4, thin layer chromatography is differentiated: it is an amount of to get the Echinacea powder, extracts with the 60-75% alcoholic solution, puts in the 75-85 ℃ of water-bath and is concentrated into small size, take out with suction pipe, be settled to 1ml, be sample liquid, other gets Echinacea control medicinal material 1.0 grams, with method operation preparation reference substance solution.Each is an amount of to get above-mentioned two kinds of solution, puts respectively on silica gel g thin-layer plate, launches with n-butyl alcohol-glacial acetic acid-water (5: 5: 1) mixed liquor, treats to take out when developing solvent is opened up to the plate forward position, and developing solvent is volatilized naturally.Observe down chromatoplate at ultra-violet lamp (365nm), in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show identical blue-fluorescence speckle.
Method 1,4 two kind of qualitative identification method are suitable for the alcohol extract of Echinacea and the compositions that contains the Echinacea alcohol extract are carried out qualitative identification equally.
In the extraction described in qualitative identification and the assay can be reflux, ultrasonic extraction.
Specific embodiments
(1). preparation said composition soft capsule
Get the Radix Panacis Quinquefolii raw material powder and be broken into coarse powder, add 8 times of amount 70% alcohol heating reflux and extract each 2 hours three times, filter, merging filtrate, being evaporated to does not have pure hiding, add the dilution of 20 times of amount deionized waters, filter D101 macroporous adsorbent resin on the filtrate, be eluted to no polysaccharide reaction with deionized water earlier, then with 70% ethanol elution till do not have a Saponin reaction, with 70% ethanol elution concentrating under reduced pressure, the concentrated solution drying of dusting, be Radix Panacis Quinquefolii extract, standby.
The Echinacea herb placed be ground into coarse powder on the pulverizer, 50% alcohol heating reflux that adds 8 times of amounts extracted 2 hours, filtered filtrate for later use, residue continues to measure 50% ethanol extraction 2 times with 6 times, each 2 hours, filter merging filtrate, concentrating under reduced pressure in the filtrate stomach concentrating under reduced pressure jar, the drying of dusting promptly gets the former powder of Echinacea extract, and is standby.
Get the raw materials ready by following prescription:
Radix Panacis Quinquefolii extract 6.4g
Echinacea extract 21.3g
PEG400 42.6g
Glycerol 8.5g
Soybean salad oil 10.6g
Water 10.6g
The supplementary material total amount is 100g, makes 100 capsules, and every capsules net content is 1g.
Take by weighing Echinacea extract, Radix Panacis Quinquefolii extract in above ratio, mix homogeneously, put and stir in the machine, start blender, slowly add the PEG400 mix homogeneously, progressively add glycerol and edible oil again, add deionized water behind the mix homogeneously again, mix rapidly, carry out homogenizing, be soft capsule content with homogenizer.
Make soft capsule peel with gelatin, G ﹠ W, soft capsule content is put fill in the soft capsule filling machine, every is adjusted into the soft capsule that contains content 1.0g, and soft capsule is put in the drying baker after the drying, be pressed into aluminum-plastic packaged in, 10 of every plates.
The effective component of product and content:
Contain among every 100g: Echinacea extract 21.3g,
Total Saponin 4270mg
Ginsenoside Rb1 480mg
Health care: immunomodulating, resisting fatigue
Suitable crowd: immunocompromised person, fatiguability person
Be not suitable for the crowd: children
Eating method and amount: every day 2-3 time, each 2
Specification: 1g/ grain
(2). the qualitative identification of some composition or component and assay in the said composition soft capsule
The total Saponin of functional component, Ginsenoside Rb in the product
1Content should meet following provisions: total Saponin (in Radix Ginseng soap Re) 〉=40000mg/kg; Ginsenoside Rb1 〉=4000mg/kg.
1. the content of ginsenoside in the spectrophotometry product
The formulation of standard curve: get ginsenoside Re's standard substance, be mixed with the standard solution that concentration is 1g/L; get standard solution 0,20,40,60,80,100 μ l (being equivalent to 0,20,40,60,80,100 μ g); be settled to 1.0ml with 70% ethanol respectively; the accurate vanillin alcoholic solution 0.5ml that adds; 80% sulfuric acid solution 5ml; shake up; put in 60 ℃ ± 1 ℃ water-bath heating 10 minutes; take out and put into ice bath cooling 15 minutes immediately; carry out colorimetric with the 1cm cuvette, in 544nm place mensuration trap, with trap to concentration return regression equation.
The mensuration of sample: 5 of sample thiefs, take out content, mix homogeneously, precision takes by weighing content 0.5g, puts in the 100ml volumetric flask, adds the 50ml water dissolution, ultrasonic oscillation extraction 20min, it is standby to be settled to 100ml again, and this is a testing sample solution.
Get above-mentioned sample solution 0.5ml, cross macroporous resin column (this post has been used acetone, and water wash purifies), after sample liquid passes through fully, use 2.5ml water, divide 4 times the drip washing pillar, blow away moisture content with rubber pipette bulb, with 70% ethanol 1.0ml eluting Radix Ginseng total saponins, collect eluent, stand-by.
With the accurate vanillin alcoholic solution 0.5ml that adds of above-mentioned eluent, 80% sulfuric acid solution 5ml shakes up, put in 60 ℃ ± 1 ℃ water-bath and heated 10 minutes, take out and put into ice bath cooling 15 minutes immediately, carry out colorimetric with the 1cm cuvette, measure trap in the 544nm place, with regression equation calculation content.
Content range: in every gram capsule 's content, the content 〉=42.7mg of total Saponin.
2. high-efficient liquid phase technique is measured the content of the ginsenoside Rb1 in the product
Chromatographic condition: chromatographic column: 0DS post (5m, 4.6 * 250nm); Mobile phase: acetonitrile-0.05% phosphoric acid solution (99: 400), flow velocity: 1ml/min detects wavelength: 203nm.
The mensuration of standard curve: precision takes by weighing ginsenoside Rb1's reference substance 10.0mg, with mobile phase dissolving and be settled in the 10ml volumetric flask, is reference substance solution.Draw reference substance solution 2 μ l, 4 μ l, 6 μ l, 8 μ l, 10 μ l sample introductions respectively, measure the data obtain peak area, with peak area to concentration return regression equation.
The mensuration of sample: 5 of sample thiefs, take out content, mix homogeneously, precision takes by weighing content 0.5g, puts in the 10ml volumetric flask, uses water dissolution, and standardize solution, draw lower floor's water, filter, get filtrate 5ml, put in the separatory funnel, with water saturated n-butanol extraction 3 times, each 10ml, merge the n-butyl alcohol phase, decompression and solvent recovery is to doing the residue dissolve with methanol, filter, filtrate is put in the 10ml volumetric flask, uses methanol constant volume, crosses the microporous filter membrane of 0.45 μ m, the accurate 20 μ l that draw, inject chromatograph of liquid, measure, by the content of ginsenoside Rb1 in the regression equation calculation sample.
Content range: content 〉=54.8mg of ginsenoside Rb1 in every gram capsule 's content.
3. the qualitative identification of Echinacea extract in the capsule
(1). the color reaction of Echinacea: get the content of 1 capsules, put in the test tube, add water 10ml, 70 ℃ of heating extraction 10 minutes are drawn the solution 2ml of lower floor and are placed test tube.Add 3 of α-naphthols alcoholic solution, shake up the tailing edge tube wall and slowly add concentrated sulphuric acid 1ml, between sulfuric acid layer and test liquid layer, occur tangible purplish red colour circle at the interface.
(2). the thin layer chromatography of Echinacea: 10 of sample thiefs, take out content, take by weighing 5.0g, with 50ml deionized water heating extraction, take off a layer solution 40ml, filter, put in the separatory funnel, use chloroform extraction 3 times, each 20ml, discard chloroform solution, water layer is transferred pH3.0, reuse ethyl acetate extraction 3 times with dilute hydrochloric acid solution (1-3ml), each 20ml, combined ethyl acetate liquid, the reclaim under reduced pressure ethyl acetate is to doing, residue is need testing solution with methanol 2ml dissolving.The methanol solution for preparing caffeic acid, chlorogenic acid concentration respectively and be 2.0mg/ml is product solution in contrast.With sample liquid 20 μ l, each 10 μ l of reference substance solution, point with the solution 10ml of the lower floor expansion of ethyl acetate-formic acid-water (10: 1: 9), takes out on the thin layer of silica gel G, fling to developing solvent, with iodine colour developing, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color, be caffeic acid and the chlorogenic acid that detects in the sample.
(3), the qualitative identification of Echinacea
1, gets Echinacea powder 0.5g, put in the triangular flask, add 70% ethanol 30ml, placed 8 hours after the jolting, filter, must get filtrate 2ml, put in the test tube, add 1～2 of 1% ferrous chloride test solution, promptly produce dark blue precipitate for examination filtrate.
2, get above-mentioned filtrate 1ml, place in vitro, add 3 of α-tea phenol alcoholic solution, shake up the tailing edge tube wall and slowly add concentrated sulphuric acid 1ml and between sulfuric acid layer and test liquid layer, occur tangible purplish red colour circle at the interface.
3, get this product powder 1g, put in the round-bottomed flask, add water 50ml hot reflux 1 hour, filter, get filtrate 5ml, add ethanol and make and contain the alcohol amount and reach 80%, jolting was left standstill 20 minutes, filtered, with the washing precipitation of 80%L alcohol, get the acclimatization thing, use water dissolution, it is a small amount of to add hydrochloric acid, and the boiling water bath heating was transferred pH＞7 with sodium hydroxide test solution after 30 minutes, and it is a small amount of to add Fehling solution, heating is several minutes on the boiling water bath, promptly produces brick-red precipitation.
4, thin layer chromatography is differentiated: get this product powder 1.0 grams, with 70% alcoholic solution reflux, extract,, put in the 75-85 ℃ of water-bath and be concentrated into about 1ml, take out with suction pipe, be settled to 1ml, be sample liquid, other gets control medicinal material 1.0 grams, with method operation preparation reference substance solution.Get each 20 μ l of above-mentioned two kinds of solution, put respectively on silica gel g thin-layer plate, launch, treat to take out when developing solvent is opened up to the plate forward position, developing solvent is volatilized naturally with n-butyl alcohol-glacial acetic acid-water (5: 5: 1) mixed liquor.Observe down chromatoplate at ultra-violet lamp (365nm), in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show identical blue-fluorescence speckle.
Compositions antifatigue effect test report
1. material and method
1.1 sample: said composition soft capsule (by the described method preparation of the application).
1.2 laboratory animal: select the Male Kunming strain mice that laboratory animal portion of Chinese Medical Sciences University provides (approval number the Liao Dynasty real moving the: No. 007) for use, body weight 18-22g.
1.3 dosage is selected: it is 6.0g/60kg.bw.d that manufacturer's recommended adult (body weight is in 60kg) takes in the approved product amount, recommended 10,20 and 30 times of adult's intake filling stomach dosage is set in minimum day with producer, 3.0s/kg.bw.d promptly 1.0,2.0,, each dosage group is with distilled water diluting, distilled water is made blank, each dosage group mouse stomach amount is 0.2mL/1og.bw.d, continuous irrigation stomach 30 days.
1.4 experimental technique:
1.4.1 swimming with a load attached to the body test
After last is tried thing 30min, put mice and swim in the case, the about 30cm of the depth of water, water temperature (25 ± 0.5) ℃, the load sheet lead of 5% body weight of Mus root of the tail portion in swimming.The record mice is from the extremely dead time of swimming beginning, as mice swimming time (min).
1.4.2 serum urea nitrogen determination
After last was tried thing 30min, the 90min that swims in the water of temperature for (30 ± 0.5) ℃ plucked the eyeball blood sampling, carried out urea nitrogen content and measured (test kit: give birth to biological worker in Beijing: journey High-tech company defends the accurate word D-81-8 of medicine number).
1.4.3 hepatic glycogen is measured:
The execution mice was got liver after last was tried thing 30min, after the normal saline rinsing, blot with filter paper, accurately take by weighing liver 75mg, every pipe adds the aqueous slkali that 225 μ L test kits provide, seal the mouth of pipe with the book film, prick an air-vent, boiling water bath 20 minutes, cooling jin adds the 7.2mL distilled water, fully mixing is pressed the operation of reagent description, (test kit: Nanjing is built up bio-engineering research and provided), carry out colorimetric determination with 722 spectrophotometric juice at the 620nm wavelength, the formula conversion hepatic glycogen that by specification provides.
1.4.4 blood lactic acid is measured
Last is tried blood sampling 20 μ L behind the thing 30min, 10min again swims in the water of temperature for (30 ± 0.5) ℃, respectively at (swimming back 0min) and swimming back rest 30min 20 μ L that respectively take a blood sample immediately after the swimming, adopt the blood lactic acid value of three time points more than the bio-sensing analysis-e/or determining that the Shandong Province academy sciences Biology Research Institute produces.(hemolytic agent and mensuration liquid provide by this institute).
1.5 experimental data is added up with the t check.
2. result
2.1 said composition is to the influence of mice swimming with a load attached to the body time and mice body weight
Table 1. said composition is to the influence of mice swimming with a load attached to the body time and mice body weight
| Dosage group (g/kg.bw) | Number of animals (only) | Starting weight (g) | Eventually heavy (g) | Weightening finish (g) | P | Swimming time (min) | p |
| Blank 1.0 2.0 3.0 | 10 10 10 10 | 19.6±1.1 19.1±1.0 19.4±0.8 19.2±1.1 | 32.9±3.7 31.8±3.5 31.7±2.6 32.2±3.5 | 13.4±3.5 12.7±3.3 12.4±2.5 13.0±3.1 | --- 0.6110 0.4660 0.7380 | 4.02±3.77 5.27±2.17 10.66±6.36△△ 6.34±4.80 | --- 0.5430 0.0024 0.2620 |
P: each experimental group and blank group are relatively
△ △ and blank group be P＜0.01 relatively
By table 1 as seen, per os gave said composition 30 days, each dosage group mice weightening finish is compared there was no significant difference (P＞0.05) with the blank group, middle dosage group swimming time is longer than the blank group, and difference has utmost point significance (P＜0.01), illustrates that said composition can prolong the mice swimming with a load attached to the body time and the mice weightening finish is not had influence.
2.2 said composition is to the influence of mice serum blood urea nitrogen and mice body weight
Table 2. said composition is to the influence of mice swimming back serum urea nitrogen and mice body weight
| Dosage group (g/kg.bw) | Number of animals (only) | Starting weight (g) | Eventually heavy (g) | Weightening finish (g) | P | Serum urea nitrogen (moL/L) | p |
| Blank 1.0 2.0 3.0 | 10 10 10 10 | 19.2±1.2 19.8±1.4 19.8±1.3 19.3±1.1 | 32.2±1.6 31.9±2.3 32.9±1.7 31.8±1.1 | 13.1±2.3 12.1±2.7 13.0±2.5 12.6±2.8 | --- 0.3830 0.9390 0.6640 | 9.35±1.22 7.07±0.86△△ 6.30±0.67△△ 5.99±0.69△△ | --- 0.0000 0.0000 0.0000 |
The p value: each experimental group and blank group are relatively
△ △ compares P＜0.01 with the blank group
By table 2 as seen, per os gives the said composition 30 days of mice various dose, each dosage group mice weightening finish is compared there was no significant difference (P＞0.05) with the blank group, high, medium and low dosage group mice swimming back serum urea nitrogen all is lower than the blank group, and with blank group comparing difference highly significant difference (P＜0.01) is arranged, illustrate that said composition has the effect of the rising that suppresses mice swimming back serum urea nitrogen and the mice weightening finish is not had influence.
2.3 said composition is to the influence of Mouse Liver glycogen content and mice body weight
Table 3. said composition is to the influence of Mouse Liver glycogen content and mice body weight
| Dosage group (g/kg.bw) | Number of animals (only) | Starting weight (g) | Eventually heavy (g) | Weightening finish (g) | P | Hepatic glycogen (mg/g hepatic tissue) | p |
| Blank 1.0 2.0 3.0 | 10 10 10 10 | 20.0±1.3 18.9±0.8 19.8±1.1 19.5±1.1 | 32.7±3.4 31.7±3.2 32.7±2.8 32.3±2.6 | 12.7±2.8 12.8±3.1 13.0±2.8 12.8±2.8 | --- 0.9940 0.7550 0.9300 | 45.31±11.33 42.10±7.85 46.43±9.41 44.65±8.82 | --- 0.4530 0.7910 0.8770 |
P value: be experimental group and the comparison of blank group
The said composition visible by table 3, that per os gives the mice various dose 30 days, each dosage group mice weightening finish is compared there was no significant difference (P＞0.05) with the blank group, each dosage group hepatic glycogen content is compared difference and is not seen significance there is (P＞0.05) with the blank group, illustrate that said composition there is no influence to the effect and the mice body weight of hepatic glycogen deposit.
2.4 said composition is to the influence of mice blood lactic acid value and mice body weight
Table 4. said composition is to the influence of mice swimming bleeding from anus lactic acid lift-off value * and mice body weight
| Dosage group (g/kg.bw) | Number of animals (only) | Starting weight (g) | Eventually heavy (g) | Weightening finish (g) | P | Before the swimming (mmoL/L) | Swimming back 0min (mmoL/L) | Lift-off value (mmoL/L) | p |
| Blank 1.0 2.0 3.0 | 10 10 10 10 | 19.5±1.2 19.6±1.1 19.6±1.2 19.0±1.0 | 32.3±2.6 31.9±3.2 32.4±3.9 31.5±3.4 | 12.8±2.5 12.3±3.6 12.8±3.1 12.4±3.3 | --- 0.7570 0.9940 0.817 | 3.2±1.3 3.1±1.0 2.9±1.0 3.2±1.1 | 4.8±0.8 4.0±0.9 3.7±0.6 4.2±0.7 | 1.5±1.0 0.9±0.5△ 0.8±0.4△ 1.0±0.5 | --- 0.0395 0.0111 0.0556 |
* lift-off value: difference of blood lactic acid measured value before blood lactic acid measured value of (swimming back 0min) blood sampling and the swimming immediately after the swimming
The p value: each experimental group and blank group are relatively
△ compares P＜0.05 with the blank group
By table 4 as seen, per os gives the said composition 30 days of mice various dose, each dosage group mice weightening finish is compared there was no significant difference (P＞0.05) with the blank group, in, the lift-off value of low dose group mice swimming bleeding from anus lactic acid all is lower than the blank group, and with blank group comparing difference significance (P＜0.05) is arranged, illustrate that said composition can reduce the generation of blood lactic acid when swimming and the mice weightening finish is not had influence.
Table 5. said composition is to the influence of mice swimming back rest 30min blood lactic acid reduction value *
| Dosage group (g/kg.bw) | Number of animals (only) | Swimming back 0min (mmoL/L) | Rest 30min (mmoL/L) | Reduction value (difference) (mmoL/L) | p |
| Blank 1.0 2.0 3.0 | 10 10 10 10 | 4.8±0.8 4.0±0.9 3.7±0.6 4.2±0.7 | 3.5±1.1 3.0±1.0 2.9±0.6 3.4±0.4 | 1.2±1.0 1.0±0.3 0.8±0.3 0.9±0.4 | -- 0.2960 0.1080 0.1660 |
* reduction value: the value that blood lactic acid is measured in (swimming back 0min) blood sampling immediately after the swimming is measured the poor of blood lactic acid value with swimming back rest 30min blood sampling.
The p value: each experimental group and blank group are relatively
By table 5 as seen, the reduction value of swimming back rest 30min blood lactic acid, high, medium and low dosage group is compared there was no significant difference (p＞0.05) with the blank group, illustrate that said composition do not see influence to blood lactic acid reduction value.
3 brief summaries
Per os gives mice said composition 1.0,2.0,3.0g/kg.bw.d, and continuous irrigation stomach 30 is big, can prolong the mice swimming with a load attached to the body time, the generation of serum urea nitrogen when reducing mice swimming, the generation of blood lactic acid when reducing swimming.Judge that thus said composition has antifatigue effect.
Said composition immunoregulation effect laboratory report
1. materials and methods
1.1 sample: said composition capsule.
1.2 laboratory animal: select the Kunming mouse that Norman Bethune Medical University medical experiment animal portion provides (the moving word of approval number doctor the: 10-5107) for use.Select 48 of healthy male mices, body weight 18-22g, be divided into 4 groups, every group 12, as immune one group, carry out internal organs/weight ratio pH-value determination pH, delayed allergy, Turnover of Mouse Peritoneal Macrophages is engulfed the chicken red blood cell experiment, the mensuration of half hemolysis value (HC50) and antibody-producting cell detect: 48 of Kunming kind Healthy female mices, and body weight 18-22g is divided into 4 groups, every group 12, as immune two groups, carry out carbon and clean up experiment: 48 of Kunming kind healthy male mices, body weight 18-22g, be divided into 4 groups, every group 12,, carry out inductive mouse lymphocyte transformation experiment of ConA and NK cytoactive and measure as immune three groups.
1.3 dosage is selected: said composition manufacturer's recommended amount is everyone (adult) 6.0g every day, becomes body weight for humans by 60kg, then everyone (adult) 6.0g/60kg every day body weight, i.e. 0.10g/kg body weight.With 10 times of human body recommended amounts, promptly every day, the 1.0g/kg body weight was as the middle dosage of mice, upper and lowerly respectively established a dosage group: the 3.0g/kg body weight is 30 times of the human body recommended amounts as high dose, and the 0.10g/kg body weight is the human body recommended amounts as low dosage.Each dosage group is tried thing and is prepared with distilled water, and blank is irritated stomach with distilled water approximately, and the continuous irrigation stomach is surveyed every immune indexes after 30 days.
1.4 experimental technique:
1.4.1 internal organs/weight ratio pH-value determination pH
Mice claims the dislocation of positive back to put to death, and gets spleen and thymus, removes most fascia, blots the organ surface blood stains with filter paper, weighs, and calculates spleen/body weight ratio and thymus/body weight ratio.
1.4.2 delayed allergy (the sufficient sole of the foot thickens method DTH)
Get Sanguis caprae seu ovis, normal saline washing 3 times, every Mus through lumbar injection 2% (V/V prepares with normal saline) hematocrit SRBC (2000tr/min, 10min) 0.2mL, after the sensitization 4 days, measure left back sufficient sole of the foot portion thickness, same position is measured and is averaged for three times.Then at measuring point subcutaneous injection 20% (V/V prepares with normal saline) hematocrit SRBC 20 μ L, measure left back sufficient sole of the foot portion thickness in injection back 24h, represent the degree of DTH with the difference (swelling degree of the paw) of sufficient sole of the foot thickness before and after attacking.
1.4.3ConA inductive mouse lymphocyte transformation experiment (mtt assay)
The aseptic spleen of getting places the little plate that fills an amount of aseptic Hank ' s liquid, makes cell suspension.Use Hank ' s liquid to wash 3 times, each centrifugal 10min (1000r/min).Then with cell suspension in the complete culture solution of 2mL, the living cell counting number, adjusting cell concentration is 2 * 10
6Individual/mL.Again cell suspension is divided two holes to add in 24 well culture plates, every hole 1mL, a hole adds 50 μ L ConA liquid (being equivalent to 5 μ g/mL) therein, and 5%CO237 ℃ of CO put in contrast in another hole
2Cultivate 72h in the cultivation.Cultivate and finish preceding 4h, supernatant 0.7mL is inhaled in every hole gently, adds the RPM1640 culture fluid 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.Then this liquid is moved in the cuvette colorimetric determination on 722 spectrophotometers, wavelength 570nm.Lymphocytic multiplication capacity deducts the optical density value that does not add the ConA hole with the optical density value that adds the ConA hole and represents.
1.4.4 antibody-producting cell detects (Jerne improves slide method)
Get Sanguis caprae seu ovis, normal saline washing 3 times, every Mus is through lumbar injection 2% (V/V prepares with normal saline) hematocrit SRBC0.2mL.The mice of SRBC immunity after 5 days put to death, get spleen, make cell suspension.After the agarose heating for dissolving, mix with the double Hank ' s of equivalent liquid, the packing small test tube, every pipe 0.5mL adds 10% (V/V is with the preparation of SA liquid) hematocrit SRBC 50L, splenocyte suspension 201 again in pipe.Rapidly behind the mixing, be poured on the slide of brushing the agarose thin layer, after treating that agar solidifies, fund is handed over the slide level to buckle and is placed on the horse, put incubation 1.5h in people's CO2 gas incubator, complement (1: 10) with the dilution of SA buffer joins in the slide frame groove then, behind the continuation incubation 1.5h, and counting hemolysis plaque number.
1.4.5 half hemolysis value (HC
50) mensuration
Get Sanguis caprae seu ovis, normal saline washing 3 times, every Mus carries out immunity through lumbar injection 2% (V/V prepares with normal saline) hematocrit SRBC 0.2mL.After 5 days, extract eyeball and get blood in centrifuge tube, place about 1h, solidification blood and tube wall are peeled off, serum is fully separated out, the centrifugal 10min of 2000r/min collects serum.With 400 times of serum dilutions, get 1mL directly in vitro with the SA buffer, add 10% (V/V is with the preparation of SA buffer) hematocrit SRBC 0.5mL successively, complement 1ml (pressing dilution in 1: 10) with the SA buffer.Other establishes the not control tube of increase serum (replacing with the SA buffer).After putting in 37 ℃ of waters bath with thermostatic control insulation 30min, the ice bath cessation reaction.The centrifugal 10min of 2000r/min gets supernatant 1ml, adds Dou Shi reagent 3ml.The get 10% simultaneously hematocrit SRBC 0.25mL of (V/V is with the preparation of SA buffer), add Dou Shi reagent to 4mL in another test tube, abundant mixing, place 10min after, sentence control tube in 540nm and do blankly, measure respectively and respectively manage optical density value.The amount of hemolysin is to count haemolysis value (HC
50) expression, be calculated as follows.
Sample HC
50Optical density value * extension rate during=sample optical density value/SRBC HD50
1.4.6 mice carbon is cleaned up experiment
The india ink of mouse tail vein injection 1: 3.5 dilution treats that prepared Chinese ink injects, immediately during juice.Inject behind the prepared Chinese ink 2,10min, get blood 20 μ L from the angular vein clump respectively, and it be added to 2mLNaCO
3In the solution.At 600nm wavelength place's photometry density value (OD), make blank with 722 spectrophotometers with Na2CO3 solution.Mice is put to death, get liver and spleen is weighed.Be calculated as follows phagocytic index a:
κ＝(1gOD
1-1God
2)/(t
2-t
1)
1.4.7 Turnover of Mouse Peritoneal Macrophages is engulfed chicken red blood cell experiment (half intracorporal method)
Mouse peritoneal injection 20% (V/V prepares with normal saline) hematocrit chicken red blood cell (2000r/min, 10min) suspension 1mL, interval 30min, the cervical vertebra dislocation is put to death, get peritoneal macrophage washing liquid 1mL, drip on microscope slide, put into the enamel box that is lined with wet gauze, 37 ℃ of incubator incubations of dislocation 30min.Incubate completely, rinsing in normal saline is to remove not paster cell.Dry, fix, 4% (V/V) Giemsa with methanol.Incubate completely, rinsing in normal saline is to remove not paster cell.Dry, fix with methanol, 4% (V/V) Giemsa-phosphate buffer dyeing, the rinsing of reuse distilled water is dried.The oil mirror is counting down, and every counting 100 macrophages are calculated as follows and engulf and phagocytic index:
Phagocytic rate %=engulfs macrophage number * 100 of the macrophage number/counting of chicken red blood cell
Phagocytic index=quilt is engulfed the macrophage number of chicken red blood cell sum/counting
1.4.8NK cytoactive is measured before (lactate dehydrogenase L DH algoscopy) experiment half an hour with target cell YAC-1 cultivations of going down to posterity, and washes 3 times with Hank ' s liquid before using, using the RPMI1640 complete culture solution adjustment cell concentration that contains 10% calf serum is 4 * 10
5Individual/mL.Being tried mice draws neck to put to death, the aseptic spleen of getting, make splenocyte suspension, use Hank ' s liquid to wash 3 times, the centrifugal 10min of 1500r/min, it is resuspended that reuse 2mL contains the RPMI1640 complete culture solution of 10% calf serum, and with the blue dyeing counting of platform phenol (viable count should more than 95%), adjusting cell concentration be 2 * 10
7Individual/mL, making and imitating the target ratio is 50: 1.Get sharp each the 100 μ L of effector lymphocyte of target cell, add in U-shaped 96 well culture plates: target cell nature release aperture adds target cell and each 100 μ L of culture fluid, and the maximum release aperture of target cell adds target cell and each 100 μ L of 1%NP40; Above-mentioned every two parallel holes, 37 ℃, 5%CO of all establishing
2Cultivate 4h in the incubator, with the centrifugal 5min of 1500r/min, every hole is drawn supernatant 100L and is put in the ELISA Plate with 96 orifice plates, add LDH substrate liquid 100 μ L, reaction 10min, every then hole adds the HCL solution 30 μ L cessation reactions of 1mol, survey the OD value at microplate reader 490nm place, the NK activity is calculated as follows:
NK cytoactive %=(reacting hole OD-nature release aperture OD)/(maximum release aperture OD-nature release aperture OD) * 100
The preparation of LDH substrate liquid: sodium lactate 5 * 10
-2Mol/L, nitro tetrazolium chloride 6.6 * 10
-4Mol/L, azophenlyene dimethyl ester sulfate 2.8 * 10
-4Mol/L, oxidized form of nicotinamide-adenine dinucleotide 1.3 * 10
-3Mo1/L is dissolved in mentioned reagent the Tris-HCl (pH8.2) in liquid of 0.2mol/L.
1.5 experimental data is added up with SPSS software variance analysis method.
2 results
2.1 said composition is to the influence of mice body weight
Table 6. said composition is to immune one group of mice body weight influence
| Group | Number of animals (only) | Starting weight (g) | Eventually heavy (g) | Weightening finish (g) | P |
| Dosage group high dose group in the blank group low dose group | 12 12 12 12 | 20.2±1.3 20.1±1.1 20.5±1.1 20.0±0.9 | 36.2±3.8 38.1±4.2 36.2±5.5 36.1±4.9 | 16.0±4.7 18.1±4.3 15.6±5.9 16.1±5.2 | --- 0.333 0.841 0.978 |
The p value: each experimental group and blank group are relatively
By table 6 as seen, learn by statistics and handle, the initial body weight of mice compares there was no significant difference (p＞0.05) between basic, normal, high dosage group and blank group, and promptly the initial body weight of mice is comparatively balanced between each group.Per os gave said composition 30 days, and there was no significant difference (p＞0.05) is compared in each dosage group mice weightening finish between basic, normal, high dosage group and blank group.Be that said composition does not have influence to the mice weight gain.
Table 7 said composition is to immune two groups of mice body weight influence
| Group | Number of animals (only) | Starting weight (g) | Eventually heavy (g) | Weightening finish (g) | P |
| Dosage group high dose group in the blank group low dose group | 12 12 12 12 | 20.5±1.1 19.6±0.9 20.0±1.1 19.8±1.1 | 34.6±5.8 33.4±4.7 34.0±4.4 34.2±4.6 | 14.1±5.7 13.9±4.8 14.0±4.3 14.4±5.3 | --- 0.901 0.965 0.885 |
The p value: each experimental group and blank group are relatively
By table 7 as seen, learn by statistics and handle, the initial body weight of mice compares there was no significant difference (p＞0.05) between basic, normal, high dosage group and blank group, and promptly the initial body weight of mice is comparatively balanced between each group.Per os gave said composition 30 days, and there was no significant difference (p＞0.05) is compared in each dosage group mice weightening finish between basic, normal, high dosage group and blank group.Be that said composition does not have influence to the mice weight gain.
Table 8 said composition is to immune three groups of mice body weight influence
| Group | Number of animals (only) | Starting weight (g) | Eventually heavy (g) | Weightening finish (g) | P |
| Dosage group high dose group in the blank group low dose group | 12 12 12 12 | 20.5±0.8 20.5±1.2 20.7±0.7 19.8±1.0 | 35.8±4.4 37.8±4.9 34.5±4.7 36.1±3.6 | 15.3±4.2 17.3±5.4 13.8±4.7 16.4±3.6 | --- 0.287 0.419 0.579 |
The p value: each experimental group and blank group are relatively
By table 8 as seen, learn by statistics and handle, the initial body weight of mice compares there was no significant difference (p＞0.05) between basic, normal, high dosage group and blank group, and promptly the initial body weight of mice is comparatively balanced between each group.Per os gave said composition 30 days, and there was no significant difference (p＞0.05) is compared in each dosage group mice weightening finish between basic, normal, high dosage group and blank group.Be that said composition does not have influence to the mice weight gain.
2.2 said composition is to the influence of mice internal organs/body weight ratio
Table 9. said composition is to the influence of mice internal organs/body weight ratio
| Group | Number of animals (only) | Spleen/body weight ratio (mg/g) | P 1 | Thymus/body weight ratio (mg/g) | P 2 |
| Dosage group high dose group in the blank group low dose group | 12 12 12 12 | 5.56±1.19 5.65±0.95 5.07±0.79 5.89±1.11 | 0.830 0.250 0.436 | 3.22±0.32 3.02±0.40 3.24±0.60 3.11±0.42 | --- 0.288 0.900 0.546 |
Spleen/body weight ratio P1 value: each experimental group and blank group are relatively
Thymus/body weight ratio P2 value: each experimental group and blank group are relatively
With table 9 as seen, per os gives the said composition 30 days of mice various dose, learn by statistics and handle, its spleen/body weight ratio and thymus/body weight ratio compares there was no significant difference (P＞0.05) between basic, normal, high dosage group and blank group, promptly said composition does not have influence to the organ weight ratio value of mice.
2.3 said composition is to the mouse cell Immune Effects
2.3.1 said composition is to the influence of mice delayed allergy (DTH)
Table 10 said composition is to the influence of mice delayed allergy (DTH)
| Group | Number of animals (only) | Swelling degree of the paw (mm) | P |
| Dosage group high dose group in the blank group low dose group | 12 12 12 12 | 0.78±0.18 0.74±0.16 0.75±0.17 0.78±0.21 | --- 0.617 0.675 0.976 |
The P value: each experimental group and blank group are relatively
By table 10 as seen, per os gives the said composition 30 days of mice various dose, learn by statistics and handle, its swelling degree of the paw compares there was no significant difference (p＞0.05) between basic, normal, high dosage group and blank group, and promptly said composition does not have influence to the delayed allergy of mice.
2.3.2 said composition is to the influence of the inductive mouse lymphocyte transformation experiment of ConA
Table 11. said composition is to the influence of the inductive mouse lymphocyte transformation experiment of ConA
| Group | Number of animals (only) | Lymphopoiesis ability (OD difference) | P |
| Dosage group high dose group in the blank group low dose group | 12 12 12 12 | 0.144±0.026 0.150±0.028 0.148±0.027 0.170±0.031* | --- 0.602 0.711 0.025 |
The p value: affair experimental group and blank group be * p＜0.05 relatively
By table 11 as seen, per os gives the said composition 30 days of mice various dose, learn by statistics and handle, its lymphocytic multiplication capacity relatively has significant difference (P＜0.05) between high dose group and blank group, compare there was no significant difference (P＞0.05) between low, middle dosage group and blank group, promptly the said composition of high dose can improve the inductive mouse lymphocyte conversion capability of ConA.2.4 said composition is to the influence of humoral immunization
Table 12. said composition is to the influence of mouse antibodies cellulation number
| Group | Number of animals (only) | Hemolysis plaque number (* 10 3Full spleen) | P |
| Dosage group high dose group in the blank group low dose group | 12 12 12 12 | 222.3±58.8 251.8±69.5 282.8±58.4* 269.3±80.6 | --- 0.289 0.033 0.094 |
The p value: each experimental group and blank group be p＜0.05 relatively
By table 12 as seen, per os gives the said composition 30 days of mice various dose, learn by statistics and handle, its antibody-producting cell number relatively has significant difference (p＜0.05) between middle dosage group and blank group, low, high dose entangles and the blank group between there was no significant difference (p＞0.05) relatively, promptly in the said composition of dosage can improve the antibody-producting cell number of mice.
Table 13. said composition is to mice half hemolysis value HC
50Influence
| Group | Number of animals (only) | Sample HC 50 | P |
| Dosage group high dose group in the blank group low dose group | 12 12 12 12 | 172±50 187±48 185±44 169±33 | --- 0.428 0.478 0.859 |
The p value: each experimental group and blank group are relatively
By table 13 as seen, per os gives the said composition 30 days of mice various dose, learn by statistics and handle, its half hemolysis value compares there was no significant difference (p＞0.05) between basic, normal, high dosage group and blank group, and promptly said composition does not have influence to the half hemolysis value of mice.
2.5.1 said composition is cleaned up the influence of function to mouse monokaryon-macrophage carbon
Table 14. said composition is cleaned up the influence of function to mouse monokaryon-macrophage carbon
| Group | Number of animals (only) | Phagocytic index (a) | P |
| Dosage group high dose group in the blank group low dose group | 12 12 12 12 | 6.35±1.19 5.69±1.46 6.35±1.12 6.69±1.43 | --- 0.226 0.989 0.519 |
The P value: each experimental group and blank Liu are according to group relatively
By table 14 as seen, per os gives the said composition 30 days of mice various dose, learn by statistics and handle, its carbon is cleaned up function and compare there was no significant difference (P＞0.05) between basic, normal, high dosage group and blank knob, and promptly said composition is cleaned up function to the carbon of mouse monokaryon-macrophage does not have influence.
Table 15. said composition is engulfed the influence of the very thin born of the same parents' of chicken ability to mouse macrophage
| Group | Number of animals (only) | Phagocytic rate (%) | P 1 | Phagocytic index | P 2 |
| Dosage group high dose group in the blank group low dose group | 12 12 12 12 | 38±10 37±10 40±8 38±10 | --- 0.708 0.598 0.843 | 0.46±0.09 0.44±0.10 0.48±0.07 0.45±0.10 | --- 0.576 0.576 0.771 |
Phagocytic rate (%) p
1Value: each experimental group and blank group be phagocytic index P relatively
2Value: each experimental group and blank group are relatively
By table 15 as seen, per os gave the mice said composition 30 days, learn by statistics and handle, its phagocytic rate relatively there are no significant between basic, normal, high dosage group and blank group difference (P＞0.05), its phagocytic index relatively there are no significant between basic, normal, high dosage group and blank group difference (p＞0.05), promptly said composition ability that mouse macrophage is engulfed chicken red blood cell does not have influence.
2.6 said composition is to the active influence of NK cells in mice
Table 16 said composition is to the active influence of NK cells in mice
| Group | Number of animals (only) | NK cytoactive (%) | P 2 |
| Dosage group high dose group in the blank group low dose group | 12 12 12 12 | 16.5±2.9 16.5±3.2 16.1±2.1 16.7±3.0 | --- 0.974 0.760 0.875 |
The p value: each experimental group and blank group are relatively
By table 16 as seen, per os gives the said composition 30 days of mice various dose, learn by statistics and handle, its NK cytoactive low, in, compare there was no significant difference (p＞0.05) between high dose group and blank group, promptly said composition does not have influence to the NK cytoactive of mice.
3 brief summary per os give the said composition 30 days of mice various dose, can strengthen the inductive mouse spleen lymphocyte conversion capability of conA, increase the antibody-producting cell number of mice; The body weight gain of mice, internal organs/body weight ratio, delayed allergy, half hemolysis value, the function that monokaryon-macrophage carbon is cleaned up, ability and the NK cell activity that peritoneal macrophage is engulfed chicken red blood cell there is not influence.Judge that thus said composition has immunoregulation effect.
Claims (4)
1, a kind of pharmaceutical composition with immunomodulating and anti-fatigue effect is characterized in that this pharmaceutical composition is to make as follows:
Get the Radix Panacis Quinquefolii raw material powder and be broken into coarse powder, add 8 times of amount 70% alcohol heating reflux and extract each 2 hours three times, filter, merging filtrate, being evaporated to does not have pure hiding, add the dilution of 20 times of amount deionized waters, filter, D101 macroporous adsorbent resin on the filtrate is eluted to no polysaccharide reaction with deionized water earlier, then with 70% ethanol elution till do not have a Saponin reaction, with 70% ethanol elution concentrating under reduced pressure, the concentrated solution drying of dusting is Radix Panacis Quinquefolii extract;
The Echinacea herb placed be ground into coarse powder on the pulverizer, 50% alcohol heating reflux that adds 8 times of amounts extracted 2 hours, filter, filtrate for later use, residue continues to measure 50% ethanol extraction 2 times with 6 times, each 2 hours, filter merging filtrate, concentrating under reduced pressure in the filtrate stomach concentrating under reduced pressure jar, the drying of dusting promptly gets the former powder of Echinacea extract;
Get the raw materials ready by following prescription: Radix Panacis Quinquefolii extract 6.4g, Echinacea extract 21.3g, PEG400 42.6g, glycerol 8.5g, soybean salad oil 10.6g, water 10.6g
Take by weighing Echinacea extract, Radix Panacis Quinquefolii extract in above ratio, mix homogeneously, put and stir in the machine, start blender, slowly add the PEG400 mix homogeneously, progressively add glycerol and edible oil again, add deionized water behind the mix homogeneously again, mix rapidly, carry out homogenizing, be soft capsule content with homogenizer;
Make soft capsule peel with gelatin, G ﹠ W, soft capsule content is put fill in the soft capsule filling machine, every is adjusted into the soft capsule that contains content 1.0g, soft capsule is put in the drying baker after the drying, be pressed into aluminum-plastic packaged in, 10 of every plates are made 100 capsules;
Contain Echinacea extract 21.3g among every 100g, total Saponin 4270mg, ginsenoside Rb1 480mg.
2, the method for quality control of pharmaceutical composition as claimed in claim 1 is characterized in that the wherein total Saponin of functional component, the Ginsenoside Rb of compositions
1Content assaying method as follows: ginsenoside's content in a. spectrophotometry compositions
The formulation of standard curve: with 70% alcoholic solution of ginsenoside Re's standard substance, the accurate vanillin alcoholic solution 0.5ml that adds, 80% sulfuric acid solution 5ml, shake up, put when heating develops the color to color stability in 60 ℃ ± 1 ℃ water-bath, taking-up is put into ice bath immediately and is cooled off colorimetric, measure trap in the 544nm place, with trap to concentration return regression equation;
The mensuration of sample: compositions is with water extraction, and the sample solution that obtains is an amount of, passes through macroporous resin column, with 70% ethanol elution Radix Ginseng total saponins, collect eluent, with accurate vanillin alcoholic solution 0.5ml, the 80% sulfuric acid solution 5ml of adding of eluent, shake up, put in 60 ℃ ± 1 ℃ water-bath and heat, take out behind the color stability and put into ice bath immediately and cool off colorimetric, measure trap in the 544nm place, in the content of ginsenoside of regression equation calculation with the ginsenoside Re;
B. high-efficient liquid phase technique is measured the content of the ginsenoside Rb1 in the product
Chromatographic condition: chromatographic column: ODS post: 5m, 4.6 * 250nm; Mobile phase: acetonitrile-0.05% phosphoric acid solution is with the mixed solvent of 99: 400 volume proportion, and flow velocity: 1ml/min detects wavelength: 203nm;
The mensuration of standard curve: an amount of with ginsenoside Rb1's reference substance, with mobile phase dissolving and standardize solution, be reference substance solution; Draw reference substance solution different volumes sample introduction respectively, measure the data obtain peak area, with peak area to concentration return regression equation;
The mensuration of sample: it is an amount of that precision takes by weighing compositions, uses water dissolution, and water intaking solution filters, filtrate is an amount of, uses water saturated n-butanol extraction, merges the n-butyl alcohol phase, decompression and solvent recovery is to doing, and the residue dissolve with methanol filters, filtrate is used methanol constant volume, cross the microporous filter membrane of 0.35-0.50 μ m, the accurate absorption in right amount injected chromatograph of liquid, in the data of 203nm mensuration peak area, by the content of ginsenoside Rb1 in the regression equation calculation sample.
3, the method for quality control of pharmaceutical composition as claimed in claim 1 is characterized in that the Echinacea in the compositions, or as follows to the method for Echinacea 40-70% ethanol extraction qualitative identification:
A. sample thief is an amount of, with water extraction, draws aqueous solution and places test tube in right amount, adds 3 of alpha-Naphthol alcoholic solution, shakes up the tailing edge tube wall and slowly adds concentrated sulphuric acid 1ml, occurs tangible purplish red colour circle between sulfuric acid layer and test liquid layer at the interface;
B. sample thief is an amount of, with water extraction, water intaking solution is an amount of, filters, with chloroform extraction, discard chloroform solution, water layer transfers to pH3.0 with dilute hydrochloric acid solution, the reuse ethyl acetate extraction, the combined ethyl acetate extract, the reclaim under reduced pressure ethyl acetate is to doing, and residue dissolves in right amount with methanol, is need testing solution; Preparation caffeic acid, chlorogenic acid concentration are the methanol solution product solution in contrast of 1.0-2.0mg/ml; Each is an amount of to get need testing solution and reference substance solution, point is on the thin layer of silica gel G, launch with lower floor's solution of ethyl acetate-formic acid-water by the mixed solvent of 10: 1: 9 volume ratios, take out, fling to developing solvent, with the iodine colour developing, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
C. sample thief is an amount of, places a few hours after adding the jolting of 60-75% ethanol, filters, and gets filtrate 2ml, adds 1～2 of 1% ferric chloride test solution, promptly produces dark blue precipitate;
D. sample thief is an amount of, extract with the 60-75% alcoholic solution, put in the 75-85 ℃ of water-bath and be concentrated into small size, take out with suction pipe, be settled to 1ml, be sample liquid, other gets Echinacea control medicinal material 1.0 grams, and with method operation preparation reference substance solution, each is an amount of to get above-mentioned two kinds of solution, put on silica gel g thin-layer plate respectively, after of the mixed liquor expansion of n-butyl alcohol-glacial acetic acid-water, under ultra-violet lamp 365nm, observe chromatoplate, in the test sample chromatograph by 5: 5: 1 volume ratios, with the corresponding position of reference substance chromatograph on, show identical blue-fluorescence speckle.
4, compositions as claimed in claim 1 has the medicine or the Application in Food of immunomodulating and anti-fatigue effect in preparation.
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| CNB031192211A CN1251697C (en) | 2003-03-05 | 2003-03-05 | Composition with functions of regulating immunity and resisting fatigue and its prepn, use and quality control method |
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| CN104705634A (en) * | 2013-12-11 | 2015-06-17 | 余邦 | Lucid ganoderma healthcare product for protecting health |
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