CN103211298A - Application of dendrobium officinale polysaccharides in preparation of harm-reducing cigarettes - Google Patents
Application of dendrobium officinale polysaccharides in preparation of harm-reducing cigarettes Download PDFInfo
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Abstract
The invention relates to application of dendrobium officinale polysaccharides in preparation of harm-reducing cigarettes. Harm reduction means reducing risks of lung inflammation caused by smoking. The harm-reducing cigarettes are prepared by dissolving the dendrobium officinale polysaccharides by utilizing water, evenly spraying the dendrobium officinale polysaccharides on cut tobaccos, naturally airing the cut tobaccos, and then rolling the cigarettes according to regular cigarette rolling technology. The application of dendrobium officinale polysaccharides in preparation of harm-reducing cigarettes can reduce the occurrence rate of lung inflammation caused by smoking and reduce expression of kinase beta (IKKbeta) of a lung tissue nuclear factor Kappa B(NF-kB) inhibitor caused by smoking.
Description
Technical field
The present invention relates to use the processing of organic substance, be specifically related to adopt the processing of Dendrobium officinale polysaccharide tobacco product tobacco product.
Technical background
Tobacco harm has become one of the most serious common problem in the world today, also is the greatest problem that human health faced.At present, the whole world has 1,100,000,000 smokers, causes nearly 5,000,000 these evitable death of example every year." Chinese harmfulness of smoking statement-of-health " also pointed out, China is maximum in the world tobacco producing country and country of consumption, and smoking is particularly serious to people's health affected.China's smoking population exceedes 300,000,000, and other the 7.4 hundred million non-smoking crowds that have an appointment suffer hazards of second-hand smoke, annual surpasses 1,000,000 because of death toll due to the smoking relevant disease.Relevant medical research shows that smoking is the behavior hazard factor of multiple illness such as cardiovascular and cerebrovascular disease, cancer, chronic obstructive pulmonary disease, and smoking has become No. second global killer after hypertension.Have data to show, the lung cancer morbidity rate of long-term smoker is higher 10 times to 20 times than the non-smoker, and epidemiology survey shows that smoking is one of important paathogenic factor of lung cancer, and about 85% is caused by smoking in the lung cancer death number, and smoking carcinogenesis is generally acknowledged.The several factors that has of lung cancer mediates, some researchers are research object with the mouse of Mus spretus kind system, gene by genetic technique location and inflammation, blood vessel generation, cellular replication and tumor susceptibility connects, the result shows the easier generation cancer of the mouse that inflammation physique is arranged, and inflammation plays significant feature to the generation development that promotes tumour.Be exposed in the cigarette and can open Ik beta kinase (IKK β) signal path, promote pulmonary inflammatory generation.Recently studies show that IKK β/NF-kB signal path is in the sustained activation state in non-small cell lung cancer (NSCLC) cell, and regulatory gene is transcribed generation, the development that participates in NSCLC.IKK β/NF-kB approach is confirmed in acute inflammation and cell survival, and except that inflammation, the continuous activation of the spy of IKK β/NF-kB approach has also promoted cancer, we can say that IKK β/NF-kB approach is important molecule tie between inflammation and the tumour.The gene target experiment has proved that IKK β activates the necessary IKK subunit of NF-kB.Previously the understanding of getting in touch to inflammation and tumour is that mainly various inflammatory cells are in inflammatory mediator, and IKK β/NF-kB approach promotes that from inflammation tumour takes place to provide a part clue with development two aspects, and complicated relation focuses on this signal transduction novel targets of IKK β between the two.Suppress IKK β specifically, for its prophylactic treatment inflammation correlation tumour provides new strategy and approach.
Smoking is harmful to your health though cigarette package is expressed, and government is great number button tax also, can not limit the increase of number of smokers.Trace it to its cause, smoking has become a kind of socialization cultural exchanges.Cigarette and wine, tea are the same, all can coordinate interpersonal relationships.Current society, especially man, cigarette has become the crucial media of its mediation interpersonal relationships." tea hand over lay Buddhist ", " wine knot chivalrous person ", tea, wine are as the thing of China's tradition communication, but the communication of cigarette is also ubiquitous, and " the meeting open-top steamed bread of meeting is passed a cigarette " increased social atmosphere, talk enjoyment, more loosened spirit.Smoking is not only the thing of sucking, especially a kind of social activityization carrier.Smoking is harmful to your health has been a undisputable fact.Raising along with the national life level, the enhancing of people's health care consciousness, how to reduce the harm of tobacco, make the smoker can reduce harm to health again to greatest extent when enjoying smoking enjoyment, China's tobacco business is mentioned important agenda to " national interests and consumer's interests are supreme ".In order to reduce the harm of the healthy generation of smoking, developing some functional harm reduction compositions becomes the research focus as tobacco additive agent.
Publication number is that the patent application of CN10275650A discloses the application of Dendrobium officinale polysaccharide in cigarette, and the method for this application is that the dendrobium candidum dendrobium polysaccharide is separated through water-soluble, and the solution that is mixed with 0.08mg/ml-0.8mg/ml is sprayed onto on the cigarette shreds, the preparation cigarette.Above-mentioned patent application also discloses Dendrobium officinale polysaccharide has been applied in the cigarette, can make when burning and sucking cigarette the flue gas form mellow and full, and fine and smooth soften increases, and dry sensation descends, and perfume quantity increases.
Summary of the invention
Technical problem to be solved by this invention provides the new purposes of Dendrobium officinale polysaccharide, i.e. the harm reduction purposes of Dendrobium officinale polysaccharide in cigarette.
Inventor's discovery that studies for a long period of time, the cigarette that contains the iron sheet dendrobium polysaccharide can significantly be reduced the cellular inflammation factor IKK β that causes because of smoking after deliberation and be expressed, and reduces the lung inflammation rate.
Therefore, the new purposes of above-mentioned Dendrobium officinale polysaccharide specifically is that the application of Dendrobium officinale polysaccharide in preparation harm reduction cigarette is characterized in that described harm reduction causes pulmonary inflammatory risk for reducing smoking.
The concrete grammar of application of the present invention is that water dissolves Dendrobium officinale polysaccharide, and evenly is sprayed on the pipe tobacco, dries naturally, according to the cigarette technology of routine tobacco cartridge is made cigarette then; Wherein, the consumption of described Dendrobium officinale polysaccharide be in the cigarette tobacco quality 0.1%～10%.
The employing water extraction and alcohol precipitation method of the Dendrobium officinale polysaccharide in the application of the present invention, supercritical ultrasonics technology or enzymatic isolation method extract and obtain.
Above-mentioned water extraction and alcohol precipitation method is made up of following steps:
The dry stem of dendrobium candidum is pulverized, crossed 50 orders, get the dendrobium candidum powder; In the dendrobium candidum powder, be incorporated as the water of 50 times of powder qualities, decoct and extract twice, each 2 hours; Merge extracted twice liquid, be concentrated into 1/10th of original volume, the volumetric concentration that adds 4 times of volumes in concentrate is 95% ethanol, standing over night, centrifugal, take off a layer sediment, fully dissolve with an amount of water after the freeze drying, add Sevage reagent and remove albumen 6 times, add the Sevage reagent of half volume of lysate at every turn, shake up, leave standstill the 30min layering, collect lower floor's aqueous phase solution, concentrate, obtain the total polysaccharide of dendrobium candidum.
Above-mentioned supercritical ultrasonics technology is made up of following steps:
The dry stem of dendrobium candidum is pulverized, crossed 50 orders, get the dendrobium candidum powder; Solid-to-liquid ratio by 1:30 adds water in the dendrobium candidum powder, ultrasonic extraction time 1.5h under 50 ℃; 5000r/min is centrifugal to remove slag, and gets supernatant, is concentrated into 1/10th of original volume, the volumetric concentration that adds 4 times of volumes is 95% ethanol, and standing over night is centrifugal, take off layer sediment with an amount of dissolved in distilled water, add Sevage reagent and remove albumen 6 times, add the Sevage reagent of half volume of dissolved in distilled water liquid at every turn, shake up, leave standstill the 30min layering, collect lower floor's aqueous phase solution, concentrate, obtain the total polysaccharide of dendrobium candidum.
The related Sevage reagent of above-mentioned water extraction and alcohol precipitation method and supercritical ultrasonics technology is to be mixed and made into by n-butanol and the chloroform volume ratio by Zheng Ding Chun ︰ chloroform=3 ︰ 1.
Above-mentioned enzymatic isolation method is made up of following steps:
The dry stem of dendrobium candidum is pulverized, crossed 50 orders, get the dendrobium candidum powder; The dendrobium candidum powder is suspended in 10 times the benzinum, adds the hot reflux degreasing, filter, collect filter residue; Add warm water in filter residue, slowly add the cellulase solution that has activated of dendrobium candidum quality 0.6% again, stir gently, be warming up to 90 ℃ of enzymes that go out immediately behind 50 ℃ of constant temperature enzymolysis 1.5h, cold filtration is collected filter residue rapidly; Add 50 times of water in the filter residue of the enzyme that goes out, lixiviate 2h under 50 ℃ of temperature filters, and adds 25 times of water lixiviate 2h under 50 ℃ of temperature again in the gained filter residue, filters; Merge the secondary filtrate add 50 times of floodings and to add 25 times of floodings, concentrate, add absolute ethyl alcohol, make that the volumetric concentration of ethanol reaches 80% in the system, standing over night in refrigerator is filtered, and filter residue gets dendrobium polysaccharide through vacuum drying.
Harm reduction cigarette of the present invention can be reduced the pulmonary inflammatory incidence that smoking is induced, the lung tissue nuclear factor Kappa B(NF-kB that downward modulation causes because of smoking) therefore the expression of the kinase beta (IKK β) of inhibitor is induced the incidence of lung cancer for reduction because of smoking and is had positive effect.
Description of drawings
Fig. 1 is for inhaling the moisture group, of cigarettes mouse changes of weight column diagram of carrying the Dendrobium officinale polysaccharide of alcohol deposition method extraction.
Fig. 2 is for inhaling the group, of cigarettes mouse changes of weight column diagram of the Dendrobium officinale polysaccharide that contains ultrasonic extraction method.
Fig. 3 is for inhaling the group, of cigarettes mouse changes of weight column diagram of the Dendrobium officinale polysaccharide that contains the enzymatic isolation method extraction.
Fig. 4～9 are the microphoto of mouse lung histotomy, wherein, Fig. 4 is blank group, Fig. 5 is for inhaling the common cigarette group, Fig. 6 is for inhaling the group, of cigarettes that contains 0.1% Dendrobium officinale polysaccharide, Fig. 7 is for inhaling the group, of cigarettes that contains 1% Dendrobium officinale polysaccharide, and Fig. 8 is for inhaling the group, of cigarettes that contains 5% Dendrobium officinale polysaccharide, and Fig. 9 is for inhaling the group, of cigarettes that contains 10% Dendrobium officinale polysaccharide.
Figure 10 is the electrophoretogram of the expression of IKK β mRNA in the mouse lung tissue, and among the figure, mark a is the normal group mouse, and mark b is for inhaling common cigarette group mouse, and mark c adds Dendrobium officinale polysaccharide group, of cigarettes mouse for inhaling.
The specific embodiment
The extraction of the total polysaccharide of example 1(dendrobium candidum)
Method one:
The dry stem of dendrobium candidum is pulverized, crossed 50 orders, get the dendrobium candidum powder; In the dendrobium candidum powder, be incorporated as the water of 50 times of powder qualities, decoct and extract twice, each 2 hours; Merge extracted twice liquid, be concentrated into 1/10th of original volume, the volumetric concentration that adds 4 times of volumes in concentrate is 95% ethanol, standing over night, centrifugal, take off a layer sediment, fully dissolve with an amount of water after the freeze drying, add Sevage reagent and remove albumen 6 times, add the Sevage reagent of half volume of lysate at every turn, shake up, leave standstill the 30min layering, collect lower floor's aqueous phase solution, concentrate, obtain the total polysaccharide of dendrobium candidum.
Method two:
The dry stem of dendrobium candidum is pulverized, crossed 50 orders, get the dendrobium candidum powder; Solid-to-liquid ratio by 1:30 adds water in the dendrobium candidum powder, ultrasonic extraction time 1.5h under 50 ℃; 5000r/min is centrifugal to remove slag, and gets supernatant, is concentrated into 1/10th, the volumetric concentration that adds 4 times of volumes is 95% ethanol, and standing over night is centrifugal, take off layer sediment with an amount of dissolved in distilled water, add Sevage reagent and remove albumen 6 times, add the Sevage reagent of half volume of dissolved in distilled water liquid at every turn, shake up, leave standstill the 30min layering, collect lower floor's aqueous phase solution, concentrate, obtain the total polysaccharide of dendrobium candidum.
Method three:
The dry stem of dendrobium candidum is pulverized, crossed 50 orders, get the dendrobium candidum powder; The dendrobium candidum powder is suspended in 10 times the benzinum, adds the hot reflux degreasing, filter, collect filter residue; Add warm water in filter residue, slowly add the cellulase solution that has activated of dendrobium candidum quality 0.6% again, stir gently, be warming up to 90 ℃ of enzymes that go out immediately behind 50 ℃ of constant temperature enzymolysis 1.5h, cold filtration is collected filter residue rapidly; Add 50 times of water in the filter residue of the enzyme that goes out, lixiviate 2h under 50 ℃ of temperature filters, and adds 25 times of water lixiviate 2h under 50 ℃ of temperature again in the gained filter residue, filters; Merge the secondary filtrate add 50 times of floodings and to add 25 times of floodings, concentrate, add absolute ethyl alcohol, make that the volumetric concentration of ethanol reaches 80% in the system, standing over night in refrigerator is filtered, and filter residue gets dendrobium polysaccharide through vacuum drying.
Example 2
One, laboratory sample is made
The making of sample 1
Get that method one resulting Dendrobium officinale polysaccharide is dissolved in water in the foregoing description 1, the Dendrobium officinale polysaccharide aqueous solution; Then, be 0.1%, 1%, 5% and 10% the made Dendrobium officinale polysaccharide aqueous solution evenly being sprayed on commercial Yuxi, Yunnan board cigarette cut tobacco of tobacco quality in the cigarette by the consumption of Dendrobium officinale polysaccharide respectively, dry naturally; At last, according to the cigarette technology of routine tobacco cartridge is made cigarette, the content that obtains Dendrobium officinale polysaccharide is respectively 0.1%, 1%, 5% and 10% cigarette.
The making of sample 2
Get that the resulting Dendrobium officinale polysaccharide of method two is dissolved in water in the foregoing description 1, the Dendrobium officinale polysaccharide aqueous solution; Then, be 0.1%, 1%, 5% and 10% the made Dendrobium officinale polysaccharide aqueous solution evenly being sprayed on commercial Yuxi, Yunnan board cigarette cut tobacco of tobacco quality in the cigarette by the consumption of Dendrobium officinale polysaccharide respectively, dry naturally; At last, according to the cigarette technology of routine tobacco cartridge is made cigarette, the content that obtains Dendrobium officinale polysaccharide is respectively 0.1%, 1%, 5% and 10% cigarette.
The making of sample 3
Get that method three resulting Dendrobium officinale polysaccharides are dissolved in water in the foregoing description 1, the Dendrobium officinale polysaccharide aqueous solution; Then, be 0.1%, 1%, 5% and 10% the made Dendrobium officinale polysaccharide aqueous solution evenly being sprayed on commercial Yuxi, Yunnan board cigarette cut tobacco of tobacco quality in the cigarette by the consumption of Dendrobium officinale polysaccharide respectively, dry naturally; At last, according to the cigarette technology of routine tobacco cartridge is made cigarette, the content that obtains Dendrobium officinale polysaccharide is respectively 0.1%, 1%, 5% and 10% cigarette.
Two, pharmacology, effect experiment
Experiment one
Contain iron sheet dendrobium polysaccharide cigarette and reduce the effect of smoking inducing mouse lung inflammation incidence
1, experiment material
1.1 animal used as test
420 body weight are the healthy male mouse of kunming of 18～22g, are provided the quality certification number by Traditional Chinese Medicine University Of Guangzhou's animal center: SCXK (Guangdong) 2008-0020.
1.2 experiment reagent and instrument
Reagent: liquid nitrogen, formaldehyde, potassium bichromate, the concentrated sulfuric acid, poly-l-lysine, haematine dye liquor, eosin stain.
Instrument: microscope digital image analysis treatment system (OLYMPUS); Animal fumigation system comprises smoking machine, flue gas conveyer air mix facilities, animal smoking apparatus for development voluntarily.
2, method
2.1 animal subject and grouping thereof
With 420 body weight is the healthy male mouse of kunming of 18～22g, is divided into 14 groups at random, 30 every group.
2.2 the group of animal subject and institute's intussusception test agent thereof
The A group is blank group (not sootiness group).
The B group is for inhaling common cigarette sootiness experimental group (cigarette is commercial Yuxi, Yunnan board cigarette).
C1, C2 and C3 group are inhaled cigarette sample 1, sample 2 and the sample 3 that contains 0.1% Dendrobium officinale polysaccharide respectively.
D1, D2 and D3 group are inhaled cigarette sample 1, sample 2 and the sample 3 that contains 1% Dendrobium officinale polysaccharide respectively.
E1, E2 and E3 group are inhaled cigarette sample 1, sample 2 and the sample 3 that contains 5% Dendrobium officinale polysaccharide respectively.
F1, F2 and F3 group are inhaled cigarette sample 1, sample 2 and the sample 3 that contains 10% Dendrobium officinale polysaccharide respectively.
2.3 experimental technique
This method adopts the only exposed mode of snuffing cigarette, that is, mouse is placed in each limiter tube, and only the respiratory nasal flue gas is avoided flue gas partly to be deposited on the fur and absorbed by animal.Control the flue gas concentration and the flow of mouse nose by setting cigarette number, air mass flow.Mouse is 400mg/m with nose exposure chamber only in flue gas granule phase substance concentration
3, smoking under the air velocity 100ml/min condition.Clear up mouse cage (cleaning once with alcohol) every morning every 2 days, remove ight soil, fill up dried wood chip, check mouse health status, food ration and the amount of drinking water of every group of mouse the previous day of weighing simultaneously, and record, change new mouse grain and drinking water, feeding at regular time and quantity of every day freely absorbed in experimentation by small white mouse.Observed and write down the body weight of mouse in per 5 days.The morning 10:00～11:00 and afternoon 15:00～16:00 be the smoking time, each three cigarettes.After continuing for 5 weeks, adopt and take off cervical vertebra method execution small white mouse fast, dissect, getting lung tissue, to put into 10% neutral formalin fixing, be used to make histotomy, row H-E dyeing, the changes in microstructure of utilizing microscopic digital image analysis processing system to observe each organ at last, carry out the conventional organization pathological examination, statistics is respectively organized mouse lung inflammation incidence.
3, result
3.1 changes of weight
35 days body weight variable quantities of each group mouse are drawn out column diagram shown in Fig. 1,2 and 3.In the time of grouping because be random packet, so respectively organize the mouse average weight significant difference does not appear.By Fig. 1,2,3 as can be seen: 1, along with the prolongation of time, smoking group weight of mice trend all is slower than normal non-smoking group, and body weight all is lighter than normal non-smoking group after the off-test, illustrates that smoking is influential to weight of mice; 2, the growth of inhaling the mouse body weight that contains iron sheet dendrobium polysaccharide cigarette is all faster than the mouse that inhales the common cigarette group, and the body weight that increases after 35 days more approaches normal group, illustrates that inhaling the cigarette that contains the iron sheet dendrobium polysaccharide influences little than common cigarette to weight of mice; 3, the body weight gain of enfleuraging between the mouse of cigarette sample 1,2 and 3 is close, does not have significant difference.
3.2 mouse lung inflammation rate
What each organized mouse lung tissue pathological slice statistics inflammation incidence the results are shown in Table 1.Data statistical approach is X
2Check.
The different group mouse lung of table 1 inflammation incidence
| Group | Radix | No lung inflammation | The inflammation incidence |
| The |
30 | 18 | 40% (spontaneous pathology) |
| The |
30 | 0 | 100％ |
| The |
30 | 6 | 80％ |
| The |
30 | 9 | 70% |
| The |
30 | 15 | 50％ |
| The |
30 | 12 | 60％ |
| The |
30 | 4 | 86.7% |
| The |
30 | 7 | 76.7％ |
| The |
30 | 12 | 60％ |
| The |
30 | 15 | 50％ |
| The |
30 | 6 | 80％ |
| The |
30 | 7 | 76.7％ |
| The |
30 | 13 | 56.7％ |
| The |
30 | 13 | 56.7％ |
By table 1 data as seen, there is significant difference (p＜0.05) between the mouse lung inflammation incidence of suction common cigarette and the normal group, illustrates that smoking is very serious to the harm of lung.The mouse lung inflammation incidence of cigarette sample 1,2 and 3 of enfleuraging all has reduction than the mouse that inhales common cigarette, wherein with 5% and 10% addition group more remarkable (p＜0.05).Experimental result explanation, the harm reduction cigarette of adding Dendrobium officinale polysaccharide can reduce the incidence of the pneumonia that smoking induces.
More as can be seen, the cigarette of the Dendrobium officinale polysaccharide preparation that water extract-alcohol precipitation extracts is in that to reduce the Dendrobium officinale polysaccharide that mouse smoking lung inflammation incidence extracts than other two kinds of methods effective with the mouse lung inflammation incidence of enfleurage cigarette sample 1,2 and 3; The Dendrobium officinale polysaccharide effect that all the other two kinds of methods are extracted is close.
Experiment two
The mouse lung pathological observation of the cigarette that the Dendrobium officinale polysaccharide that suction is extracted by water extraction and alcohol precipitation method prepares
1 experiment material
1.1 animal used as test
60 body weight are the healthy male mouse of kunming of 18～22g, are provided the quality certification number by Traditional Chinese Medicine University Of Guangzhou's animal center: SCXK (Guangdong) 2008-0020.
1.2 experiment reagent and instrument
Tried cigarette sample: be the cigarette that the Dendrobium officinale polysaccharide that is extracted by water extraction and alcohol precipitation method prepares, promptly above-mentioned cigarette sample 1.
Reagent: liquid nitrogen, formaldehyde, potassium bichromate, the concentrated sulfuric acid, poly-l-lysine, haematine dye liquor, eosin stain.
Instrument: microscope digital image analysis treatment system (OLYMPUS); Animal fumigation system comprises smoking machine, flue gas conveyer air mix facilities, animal smoking apparatus for development voluntarily.
2 methods
With 60 body weight is the healthy male mouse of kunming of 18～22g, is divided into 6 groups at random, 10 every group.The A group is common cigarette sootiness experimental group for blank group (not sootiness group), B group; C group for contain 0.1% Dendrobium officinale polysaccharide cigarette, D group for contain 1% Dendrobium officinale polysaccharide cigarette, E organize for contain 5% Dendrobium officinale polysaccharide cigarette, F organizes to containing 10% Dendrobium officinale polysaccharide cigarette.
This method adopts the only exposed mode of snuffing cigarette, that is, mouse is placed in each limiter tube, and only the respiratory nasal flue gas is avoided flue gas partly to be deposited on the fur and absorbed by animal.Control the flue gas concentration and the flow of mouse nose by setting cigarette number, air mass flow.Mouse is 400mg/m with nose exposure chamber only in flue gas granule phase substance concentration
3, smoking under the air velocity 100ml/min condition.Clear up mouse cage (cleaning once with alcohol every 2 days) every morning, remove ight soil, fill up dried wood chip, check mouse health status, feeding at regular time and quantity of every day freely absorbed in experimentation by small white mouse.The morning 10:00～11:00 and afternoon 15:00～16:00 be the smoking time, each three cigarettes.After continuing for 5 weeks, employing is taken off the cervical vertebra method fast and is put to death small white mouse, dissects, and after observation organ color, the Volume Changes, gets left lung frozen and-70 ℃ of refrigerators rapidly, is used for machine-processed experimental study; It is fixing that second half right lung tissue is put into 10% neutral formalin, is used to make histotomy, row H-E dyeing, and the changes in microstructure of utilizing microscopic digital image analysis processing system to observe each organ is at last carried out the conventional organization pathological examination.Above-mentioned mouse lung histotomy under light microscope the resultant picture of imaging shown in Fig. 4-9.
3 results
Perusal mouse lung histotomy (seeing Fig. 4-9) as seen, blank group mouse lung smooth surface, the lung tissue pinkiness, the surface does not have congested, no oedema, matter is tough; Common cigarette group mouse lung tissue surface is coarse, gray, and there is hyperemia on the surface, oozes out, and matter is hard slightly; Contain different proportion Dendrobium officinale polysaccharide group, of cigarettes mouse lung tissue and approach blank group mouse, do not have congested, no oedema, the lung tissue color is than deep space Bai Zushen.
By Fig. 4-9 as seen, blank group mouse lung is organized the epithelial structure complete display, and alveolus wall no abnormality seen pathological change is not seen inflammatory reaction, and blood vessel does not have expansion.Common group of mouse lung tunica mucosa bronchiorum epithelial hyperplasia is serious, and have and come off, the tube wall proliferation of smooth muscle is obvious, alveolar septa thickens obviously, and as seen cell infiltration is arranged, and interstitial pneumonia is obvious, the alveolar major part is highly expansion, visible protein liquid of part and cellulose soak into, and pulmonary emphysema, pulmonary edema are obvious, part lumen of vessels, a matter hyperemia.It is not obvious to contain 0.1% Dendrobium officinale polysaccharide group, of cigarettes mouse lung tunica mucosa bronchiorum epithelial proliferation, does not have to come off, destroy, and the bronchial smooth muscle hyperplasia is comparatively obvious, as seen adipocyte, alveolar septa thickens comparatively obvious, and wind-puff, oedema are not obvious, but with slight bronchiectasis.It is complete to contain 1% Dendrobium officinale polysaccharide group, of cigarettes mouse lung bronchial tissue epithelial structure, and alveolar structure is more complete, no wind-puff, oedema, hyperemia, and alveolar is separated with hyperplasia, with slight interstitial pneumonia.It is complete to contain 5% Dendrobium officinale polysaccharide group, of cigarettes mouse lung bronchus structure, no wind-puff, oedema, hyperemia, and alveolar is separated with slight hyperplasia.It is complete to contain 10% Dendrobium officinale polysaccharide group, of cigarettes mouse lung bronchus structure, no wind-puff, oedema, hyperemia, and alveolar is separated with slight hyperplasia, slight interstitial pneumonia, the part blood vessel is congested on a small quantity.
Common group of cigarette mouse lung of above-mentioned suction tissue pathological slice presentation of results smog of smoking is very outstanding to the histology infringement of lung.The lung pathology section result who adds each harm reduction group, of cigarettes mouse of Dendrobium officinale polysaccharide shows that the lung inflammation pathological manifestations obviously alleviates, and illustrate that the cigarette of adding Dendrobium officinale polysaccharide can reduce the damage to lung tissue, prevents and the generation of reduction lung cancer.
Above-mentioned each experimental result shows that all the cigarette that contains the iron sheet dendrobium polysaccharide can obviously alleviate the harmful effect that smoking causes, reduces the harm that smoking causes body, protection body, the generation of prevention lung cancer.
Experiment three
The expression of IKK β mRNA in the mouse lung tissue
1 experiment material
1.1 the lung tissue of experiment mice
The lung tissue of respectively organizing mouse in the above-mentioned experiment two.
1.2 experiment reagent and instrument
Reagent: liquid nitrogen, the ultrapure total RNA rapid extraction kit of RNApure (Beijing Ai Delai bio tech ltd), universal RT-PCR kit (Beijing DingGuo ChangSheng Biology Technology Co., Ltd), IKK β primer (the English Weihe River, Shanghai Jie Ji Bioisystech Co., Ltd), agarose (Spain), Mouse β-actin(Beijing DingGuo ChangSheng Biology Technology Co., Ltd).
Instrument: 1300SERIES A2 Biohazard Safety Equipment (Thermo, Germany), LIBROR AEG-220 electronic analytical balance (SHIMADZU, Japan), TDZ5-WS centrifuge (Hunan, Changsha intelligence centrifuge instrument Co., Ltd), GL-21M high speed freezing centrifuge (Shanghai Lu Xiang instrument centrifuge instrument Co., Ltd), BCD-205TJ refrigerator (Qingdao HaiEr Co., Ltd).2400 type PCR instrument (Applied Biosystems, Singapore), UV-1800 type UV-detector (Tianjin, island, Japan), DYY-2C type electrophoresis apparatus (Liuyi Instruments Plant, Beijing).
2 methods
2.1 total RNA extracts
The abundant precooling mortar of liquid nitrogen, pestle, precooling gets final product for 3 times repeatedly, rapidly the 100mg mouse lung tissue of sharp freezing is put into the mortar of precooling, fully grind lung tissue on ice with pestle, until powdered, blow and beat a moment behind the adding 1ml lysate, collecting cell is in the EP pipe.Extract kit by RNA and illustrate that the total RNA of lung tissue is respectively organized in extraction respectively.With total RNA of obtaining ratio in 1:10, with RNase-free water dilution, promptly to draw the total RNA sample of 5ul and add among the 45ul RNase-free water, mixing is measured absorbance under 260nm, the 280nm, i.e. OD260 and OD280 with ultraviolet specrophotometer.The sample of OD260/OD280 between 1.8～2.0 can carry out Real Time-PCR.
2.2cDNA synthetic
By the operation of reverse transcription kit operation instruction.Reaction system is as follows: 5 * M-MLV Buffer2ul, dNTP Mixture0.5ul, Random6mers0.5ul, RTase M-MLV (RNase H
-) (200U/ μ l) 0.25, RNase Inhibitor (40U/ μ l) 0.25, Total RNA500ng, RNase Free dH
2O is to cumulative volume 20ul.Reverse transcription reaction condition: 42 ℃ of insulation 1h, cooled on ice behind 70 ℃ of insulation 15min.Reaction is positioned over-20 ℃ of preservations with cDNA solution after finishing.
2.3PCR detect
Primer is synthetic by the design of Invitrogen company, IKK β upstream primer 5 '-AGCTCTGGAACCTCCTGAAGA-3 ', downstream primer 5 '-AGCTCCAGTCTAGGGTCGTGA-3 ', amplified production 246bp; β-actin upstream primer 5 '-GTGGGACGACATGGAGAAAA-3 ', downstream primer 5 '-AAGGAAGGCTGGAAGAGTGC-3 ', amplified production 564bp.The PCR reaction system is as follows: above-mentioned cDNA solution 1 μ L, Taq enzyme (5U/ μ L) 0.5 μ L, 10 * PCR Buffer (Mg2+Plus), 5 μ L, Dntp (2.5mM) 4ul, β-actin upstream primer (10 μ M) 0.5 μ L, β-actin downstream primer (10 μ M) 0.5 μ L, IKK β upstream primer (10 μ M) 0.5 μ L, IKK β downstream primer (10 μ M) 0.5 μ L, dH2O (sterile purified water) is to cumulative volume 50 μ L.Reaction condition: 94 ℃, 1min; 98 ℃, 10sec; 56 ℃, 15sec; 72 ℃, 1min; 72 ℃, 10min, 35 circulations.
2.4 gel electrophoresis
With above-mentioned PCR product through 2% agarose gel electrophoresis, voltage 120V, electric current 200MA, time 40min adopts the gel imaging analysis instrument to carry out graphical analysis.
3 results
Above-mentioned gel imaging system analytical electrophoresis result as shown in figure 10.As can be seen from Figure 10, normal group mouse and inhale in the cigarette mouse lung of Dendrobium officinale polysaccharide and all do not have the expression of IKK β mRNA, and inhale the expression that obvious IKK β is arranged in the mouse lung tissue of common group of cigarette.The expression that can reduce the inflammatory factor IKK β mRNA that causes because of smoking of experimental result explanation Dendrobium officinale polysaccharide reduces the lung inflammation incidence.
Claims (2)
1. the application of Dendrobium officinale polysaccharide in preparation harm reduction cigarette is characterized in that described harm reduction causes pulmonary inflammatory risk for reducing smoking.
2. application according to claim 1, it is characterized in that the preparation method of described harm reduction cigarette is made up of following steps: water dissolves Dendrobium officinale polysaccharide, and evenly be sprayed on the pipe tobacco, naturally dry, according to the cigarette technology of routine tobacco cartridge is made cigarette then; Wherein, the consumption of described Dendrobium officinale polysaccharide be in the cigarette tobacco quality 0.1%～10%.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2013101432578A CN103211298A (en) | 2013-04-23 | 2013-04-23 | Application of dendrobium officinale polysaccharides in preparation of harm-reducing cigarettes |
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| CN2013101432578A CN103211298A (en) | 2013-04-23 | 2013-04-23 | Application of dendrobium officinale polysaccharides in preparation of harm-reducing cigarettes |
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| CN103211298A true CN103211298A (en) | 2013-07-24 |
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| CN2013101432578A Pending CN103211298A (en) | 2013-04-23 | 2013-04-23 | Application of dendrobium officinale polysaccharides in preparation of harm-reducing cigarettes |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105194346A (en) * | 2015-10-30 | 2015-12-30 | 广州中医药大学 | Traditional Chinese medicine composition with weight reducing function and preparation method of traditional Chinese medicine composition |
| WO2016019548A1 (en) * | 2014-08-07 | 2016-02-11 | 香港大学 | Pharmaceutical use of polysaccharide of dendrobium officinale |
| CN108851179A (en) * | 2018-08-07 | 2018-11-23 | 武汉纺织大学 | One kind containing Herba Dendrobii extract additive in cigarette and preparation method thereof |
| CN113308301A (en) * | 2021-06-04 | 2021-08-27 | 安徽中烟工业有限责任公司 | Dendrobium polysaccharide Amadori latent aroma spice, preparation method thereof and application thereof in cigarettes |
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| CN102715650A (en) * | 2012-06-21 | 2012-10-10 | 浙江中烟工业有限责任公司 | Application of Dendrobium officinale polysaccharide to cigarette |
| CN102987539A (en) * | 2012-12-24 | 2013-03-27 | 上饶市汇草药材研究所 | Manufacture process of dendrobium officinale cigarette |
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| JPH01117772A (en) * | 1987-10-30 | 1989-05-10 | Japan Tobacco Inc | Agent for improving flavor of tobacco |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016019548A1 (en) * | 2014-08-07 | 2016-02-11 | 香港大学 | Pharmaceutical use of polysaccharide of dendrobium officinale |
| CN105194346A (en) * | 2015-10-30 | 2015-12-30 | 广州中医药大学 | Traditional Chinese medicine composition with weight reducing function and preparation method of traditional Chinese medicine composition |
| CN105194346B (en) * | 2015-10-30 | 2020-01-31 | 广州中医药大学 | Traditional Chinese medicine composition with weight-losing effect and preparation method thereof |
| CN108851179A (en) * | 2018-08-07 | 2018-11-23 | 武汉纺织大学 | One kind containing Herba Dendrobii extract additive in cigarette and preparation method thereof |
| CN113308301A (en) * | 2021-06-04 | 2021-08-27 | 安徽中烟工业有限责任公司 | Dendrobium polysaccharide Amadori latent aroma spice, preparation method thereof and application thereof in cigarettes |
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Application publication date: 20130724 |