CN110123893A - Smoked plum extractive is preparing the application in drug or health care product for preventing and treating the death of mouth epithelial cells caused by buccal cigarette - Google Patents
Smoked plum extractive is preparing the application in drug or health care product for preventing and treating the death of mouth epithelial cells caused by buccal cigarette Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
- A61K36/736—Prunus, e.g. plum, cherry, peach, apricot or almond
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The present invention relates to smoked plum extractives to prepare the application in drug or health care product for preventing and treating the death of mouth epithelial cells caused by buccal cigarette, belongs to new tobacco products studying technological domain.Present invention discover that people, when product eat buccal cigarette, the osmotic pressure of mouth epithelial cells microenvironment increases；Stomatocyte, which is exposed in hypertonic environment, will lead to cell death；Smoked plum extractive is added in hypertonic solution can improve because of cell death caused by it.The present invention is that smoked plum extractive has excavated new purposes, is laid the foundation for the hypertonic caused mouth epithelial cells death Protective substances of Future Development.
Description
Technical field
The invention belongs in new tobacco products studying technological domain, and in particular to a kind of smoked plum extractive is used in preparation
The drug for preventing and treating mouth epithelial cells death caused by buccal cigarette or the application in health care product.
Background technique
Oral cavity is the earliest contacting foodstuff of human body and the organ for starting digestion.In oral epithelium, there is no angles at most of positions
Change layer (being compared with skin), the rete malpighii (being compared with gastrointestinal tract) also formed without mucus, therefore mouth epithelial cells are directly sudden and violent
It is exposed in external environment, when the moieties in food and medicine generate dead to epithelial cell, oral cavity discomfort occurs therewith.
Research shows that the reason of Stomatocyte death first is that the osmotic pressure of saliva of buccal cavity is received and extraneous influences to be changed.
People is about 50 mOsmol/L in the osmotic pressure of tranquillization state saliva, and intake and laboratory rodent chow after, due to food degradation and
The release of small molecule (including salt ion, glucose and amino acid etc.), intraoral osmotic pressure will rise rapidly to 600-900
MOsmol/L is about as much as 2-3 times (normal osmotic pressure is 285-295 mOsmol/L) of human body fluid normal osmotic pressure.
Buccal cigarette refers mainly to the smoke-free tobacco product chewed by oral cavity, consumed containing modes such as change, mouth containings.Consumption
It is uncomfortable that person's questionnaire survey shows that the packed buccal cigarette in part can cause oral cavity, including causes lip uncomfortable, and saliva amount increases, oral cavity
Spinosity excitation etc., to influence mouth feel of consumer when using product.Since part buccal cigarette can add sugar, salinity
Equal ingredients, form hypertonic environment so as to cause oral cavity.
Dark plum is the drying almost ripe fruit of rose department plant plum Prunus mume (Sieb.) Sieb.etZuce., is had
The effect of astringing lung-QI, puckery intestines, promotes the production of body fluid, claming ascaris.It is usually used in chronic cough of deficiency lung, protracted dysentery and diarrhea, abnormal heat is quenched one's thirst, and the ascariasis of biliary tract vomits abdominal pain.This hair
It is bright to protect its application into the death of mouth epithelial cells caused by buccal cigarette, relevant report that so far there are no.
Summary of the invention
It is an object of the present invention to solve the deficiency of the existing technology and provide a kind of smoked plum extractive in preparation for preventing and treating
The drug of the death of mouth epithelial cells caused by buccal cigarette or the application in health care product.
To achieve the above object, The technical solution adopted by the invention is as follows:
Smoked plum extractive is preparing answering in drug or health care product for preventing and treating the death of mouth epithelial cells caused by buccal cigarette
With.
It is further preferred that the buccal cigarette is packed buccal cigarette.
It is further preferred that the preparation method of the smoked plum extractive includes the following steps:
Step (1) after taking dark plum dried product to crush, is shaken with ultrapure water and is extracted；
Step (2), extracting solution use high speed freezing centrifuge, are centrifuged at 4 DEG C, leave and take supernatant, abandon precipitating；
Step (3) saves backup after supernatant freeze-drying, obtains smoked plum extractive.
It is further preferred that being extracted repeatedly with ultrapure water concussion, 1 hour every time.
It is further preferred that the dry capitulum 100g of dark plum is weighed, after crushing, respectively with 500 ml, 300 ml
Each primary with the ultrapure water concussion extraction of 200ml, each extraction time is 1 hour.
It is further preferred that the parameter of noncentricity of high speed freezing centrifuge be 16000 g, 30 minutes.
It is further preferred that freeze temperature is -30 DEG C, the time is 24 hours.
Those skilled in the art should know that the health care product includes food and drink with health care function.
Those skilled in the art should know that the present invention is not limited to the above method for the extracting method of smoked plum extractive.
There is no limit for step (1) smashed partial size by the present invention.
The dark plum dried product that the present invention uses can contain fruit stone for commercial product.
The present invention uses common NaCl and glucose (Glucose) in buccal cigarette first, is added in serum free medium,
The change of osmotic pressure, then the human oral epithelial cells hTERT-OME immortalized with the culture medium stimulation of different osmotic are measured,
In experiment, while smoked plum extractive group (smoked plum extractive of various concentration is added in hypertonic culture medium), blank group (training are set
It is support base and MTS solution, cell-free), control group (cell normally cultivated without high osmotic treatment), three multiple holes of every group of setting.
MTS reagent box is used to detect survivaling cell after stimulating a period of time.The result shows that: people is when product eat buccal cigarette, oral epithelium
The osmotic pressure of cell micro-environment increases；Stomatocyte, which is exposed in hypertonic environment, will lead to cell death；Add in hypertonic solution
Entering smoked plum extractive can improve because of its caused cell death.The present invention is that smoked plum extractive has excavated new purposes, is
Oral epithelium injury protection substance caused by Future Development is hypertonic lays the foundation.
Compared with prior art, the present invention has the advantages that:
1, it is to cause the change of saliva osmotic pressure that research of the invention, which discloses buccal cigarette to influence the major reason of oral cavity discomfort,；?
During test, osmotic pressure can simply be assessed to the detection method of impact cell by establishing one kind, passed through this method and found crow
It is dead that prunus mume extract can be used in mouth epithelial cells caused by preventing and treating buccal cigarette；
2, the present invention is that smoked plum extractive has excavated new purposes, is that mouth epithelial cells death caused by Future Development is hypertonic is protected
Shield substance lays the foundation.At the high NaCl culture medium or high glucose culture medium of 2 times of physiological osmotic pressures (580mOsmol/L)
Reason can lead to the cell death of cell about 40% for mouth epithelial cells 8-12 hours, and the smoked plum extractive of 10 mg/ml is added
Then the ratio of cell death can be reduced to 10% or less.
3, the present invention is that mouth epithelial cells death caused by smoked plum extractive directly uses middle and high infiltration in buccal cigarette is protected
In provide the foundation.Mouth containing tobacco extract and epithelial cell, which are incubated for, causes about 50% mouth epithelial cells dead for 8 hours, but adds
The ratio of cell death then can be reduced to 10% or less by the smoked plum extractive for entering 10 mg/ml.
Detailed description of the invention
Fig. 1 is tranquillization state saliva of buccal cavity osmometry result and the result using oral cavity osmotic pressure after buccal cigarette；
Fig. 2 is that hyperosmosis acts on testing result to cell death；Wherein, the side that (A) passes through addition sodium chloride in the medium
Osmotic pressure culture medium is turned up in method, after medium treatment mouth epithelial cells 12 hours of different osmotic, measures cell with MTS
Cell survival ratio is calculated after vigor；(B) osmotic pressure culture medium is turned up by the method that glucose is added in the medium, with not
After medium treatment mouth epithelial cells 12 hours of osmotic pressure, cell survival ratio is calculated after measuring cell viability with MTS；
(C) with the high sodium chloride medium treatment cell difference of twice physiological osmotic pressure (physiological osmotic pressure is 290 mOsmol/L) when
Between, after with MTS measure cell viability, calculate cell survival ratio；(D) with twice physiological osmotic pressure (physiological osmotic pressure 290
MOsmol/L high glucose medium treatment cell different time), after with MTS measure cell viability, calculate cell survival ratio
Example；
Fig. 3 is the intervention result after adding smoked plum extractive to hypertonic caused cell death；Wherein, A is to use high sodium chloride
It is that caused hypertonic culture medium obtains as a result, B be use the testing result arrived in hypertonic culture medium caused by high glucose；
Fig. 4 is after adding Flos Chrysanthemi Indici extract to the intervention result of cell death caused by mouth containing tobacco extract.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail.
It will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and it should not be regarded as limiting this hair
Bright range.In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art
Or it is carried out according to product description.Production firm person is not specified in material therefor or equipment, is that can be obtained by purchase
Conventional products.
Embodiment 1: people's saliva osmotic pressure after ingesting gum base type buccal cigarette 1 minute
(1) 25 volunteers of health have been recruited at random, and volunteer's basic condition is as follows: the age: 22-24 years old；Gender: male
Property 12, women 13；
(2) every volunteer collects its saliva in quiescent condition, and 16000rpm is centrifuged 5min, and supernatant is used for osmometry.
(3) after tranquillization state acquires saliva interval 1 hour, volunteer uses buccal cigarette (Snus Frost, beauty according to explanation
State's Camel product).
(4) whole saliva in the 1st minute collection volunteer oral cavity (period can not swallow or spue saliva) after use.
(5) saliva osmotic pressure is measured, assessment smokeless tobacco uses the influence to saliva osmotic pressure.Measurement result
As shown in Figure 1, volunteer is 42.88 mOsmol/L in the saliva osmotic pressure average value of tranquillization state.And after using buccal cigarette then
For 619.60 mOsmol/L, increase more than ten times.
These results explanation, when using buccal cigarette, oral cavity will be exposed in hypertonic microenvironment.
Embodiment 2: hyperosmosis acts on cell death and detects
(1) cell culture and passage: the human oral epithelial cells hTERT-OME of immortalization is purchased from Abmgood company, and is incubated at
Contain the MCDB151 culture medium (Sigma-Aldrich product) that percentage by volume is 1% fetal calf serum (Hyclone product)
25cm2In culture bottle.After cell grows to 80% Fusion Strain, culture solution is abandoned, washed once with PBS, 0.5-1mL is added
0.25% trypsase -0.53mM EDTA digestive juice (Sigma-Aldrich product), 37 DEG C digestion 1-5 minutes.Cell rounding
Afterwards, digestive juice is discarded.The MCDB151 culture medium containing percentage by volume for 10% fetal calf serum is added, cell, 600g is resuspended in piping and druming
Centrifugation 5 minutes, abandons supernatant, and cell is resuspended in the aforementioned MCDB151 culture medium containing percentage by volume for 1% fetal calf serum of 15 ml
In, it is passed on according to the ratio of 1:3.It changes the liquid once within every 2-3 days, half amount changes liquid, is to change into new to contain volume hundred when changing liquid
Score is the MCDB151 culture medium of 1% fetal calf serum.
(2) hypertonic solution is configured using MCDB151 serum free medium, respectively using the method for addition NaCl, Glucose
The osmotic pressure in culture medium is adjusted, the culture medium of different osmotic is carried out to cell or fixed osmotic pressure carries out different time thorn
Swash.The osmotic pressure used is 1.25 times, 1.50 times, 1.75 times, 2.00 times and 2.50 times of serum free medium.Free serum culture
The osmotic pressure of base is 290 mOsmol/L, which is also physiological osmotic pressure.
(3) hTERT-OME for being in logarithmic growth phase is inoculated in 96 orifice plates, every hole cell quantity is 8000, training
Supporting matrix product is 100 μ l, using serum-free MCDB151 culture medium.After cell adhere-wall culture 24 hours, cell culture medium is replaced
Isotonic or hypertonic culture medium to prepare in above-mentioned (2) is cultivated.
Influence of the hypertonic environment to cell survival rate is detected using the following two kinds mode:
A) with 2 times of physiological osmotic pressures (580 mOsmol/L) processing cell different time, (4 hours, 8 hours, 12 hours and 24 are small
When)；
B) it is handled cell 12 hours with different osmotic (290,363,435,580,1160 mOsmol/L).
(4) in cell culture terminal, survivaling cell is detected using the MTS reagent box of Promega, in each cell culture well
10 μ l MTS solution of middle addition, 37 DEG C are incubated for 2 hours.Measure the light absorption value in each hole 490 nm.In an experiment, while sky is set
White group (culture medium and MTS solution, cell-free), control group (cell normally cultivated without high osmotic treatment), every group of setting three
A multiple holes.
(5) experimental result is as shown in Figure 2: after adding additional sodium chloride or glucose in the medium, when cell occurs
Between and osmotic pressure rely on cell death.When fixed different osmotic medium treatment cell stage is 12 small, change culture
When base osmotic pressure, at 2 times of physiological osmotic pressures (580 mOsmol/L), that is, there is about 50% cell death.Cell draws glucose
The hyperosmosis risen is more resistant to, and the hypertonic culture medium of 4 times of glucose can cause 50% cell death.Film solid media
Osmotic pressure is that 580 mOsmol/L stimulate cell, and cell extends with stimulation time, the decline of cell survival ratio.
The preparation of 3 smoked plum extractive of embodiment
(1) dark plum 100g(dry product is weighed, includes fruit stone), after crushing, respectively with the ultrapure water of 500 ml, 300 ml and 200ml
Concussion extraction is each primary, and each extraction time is 1 hour.
(2) combined extract, 16000 g high speed refrigerated centrifuges (4 DEG C, 16000g, 30 minutes) leave and take supernatant, and it is heavy to abandon
It forms sediment.
(3) it is saved backup after supernatant freeze-drying, obtains smoked plum extractive, lyophilisation condition are as follows: -30 DEG C, 24 hours.
Intervention after the addition smoked plum extractive of embodiment 4 to hypertonic caused cell death
1. detecting cell death according to method identical in above-described embodiment 2, the condition of 2 times of physiological osmotic pressures, i.e., 580 are chosen
MOsmol/L is tested, and is set as 8 hours (sodium chloride) or 12 hours (grapes to the stimulation time of cell hTERT-OME
Sugar).
2. the smoked plum extractive of various concentration is added in hypertonic culture medium, concentration includes: 1 mg/ml, 5 mg/ml, 10
mg/ml.After cultivating above-mentioned specified time, cell survival rate is detected with MTS method.
3. experimental result (mentions as shown in figure 3, smoked plum extractive is added in the medium according to the method in embodiment 3
Take), cell death caused by can inhibiting hypertonic in dose-dependent mode, 10 mg/ml smoked plum extractives can be almost complete
It is complete to inhibit because mouth epithelial cells caused by hypertonic are dead.In isotonic situation, 5 mg/ml smoked plum extractives are not thin to cell
Cellular toxicity.In hypertonic culture medium, smoked plum extractive can play guarantor to cell in various degree in various concentration (1-10 mg/ml)
Shield effect.
Intervention after the addition smoked plum extractive of embodiment 5 to cell death caused by mouth containing tobacco extract
1. preparing mouth containing tobacco extract with MCDB151 culture medium: taking lip cigarette (Snus Frost) 1,2 ml MCDB151 are added
Culture medium (is preheated to 37 DEG C), is placed in mortar, is squeezed 4 times (interval squeezes primary for 15 seconds) with grinding pestle.After 1 min, cigarette is abandoned
Bag and its remaining content, take 16000 rpm of extract liquor to be centrifuged 10 min, spare through 0.22 μm of membrane filtration, if do not made immediately
With freezing in -80 DEG C.
2. sucking tobacco extract osmometry: to the extract in above-mentioned 1 carry out osmometry (U.S./
Wescor), osmotic pressure is measured are as follows: 785 ± 17 mOsmoL/kg.
3. detecting cell death according to method identical in above-described embodiment 2, the culture medium of cytositimulation includes: that (1) is right
According to group: serum-free MCDB151 culture medium；(2) 10 mg/ml dark plums smoked plum extractive control group: are added in MCDB151 culture medium
Extract；(3) tobacco extract stimulation group is sucked: the mouth containing tobacco extract extracted with MCDB151；(4) smoked plum extractive protection group:
10 mg/ml smoked plum extractives are added in the mouth containing tobacco extract extracted with MCDB151.Stimulation time is set as 8 hours, uses MTS
Method detects cell survival rate.
4. experimental result deposits cell as shown in figure 4, adding 10 mg/ml smoked plum extractives in MCDB151 culture medium
Work does not influence, and mouth containing tobacco extract leads to about 50% cell death, but sucking after stimulation mouth epithelial cells 8 hours
10 mg/ml smoked plum extractives are added in tobacco extract to suck cell death caused by tobacco extract with effective protection.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Claims (7)
1. smoked plum extractive is in preparing drug or health care product for preventing and treating the death of mouth epithelial cells caused by buccal cigarette
Using.
2. smoked plum extractive according to claim 1 is in preparation for preventing and treating the death of mouth epithelial cells caused by buccal cigarette
Drug or health care product in application, which is characterized in that the buccal cigarette be packed buccal cigarette.
3. smoked plum extractive according to claim 1 is in preparation for preventing and treating the death of mouth epithelial cells caused by buccal cigarette
Drug or health care product in application, which is characterized in that the preparation method of the smoked plum extractive includes the following steps:
Step (1) after taking dark plum dried product to crush, is shaken with ultrapure water and is extracted；
Step (2), extracting solution use high speed freezing centrifuge, are centrifuged at 4 DEG C, leave and take supernatant, abandon precipitating；
Step (3) saves backup after supernatant freeze-drying, obtains smoked plum extractive.
4. smoked plum extractive according to claim 3 is in preparation for preventing and treating the death of mouth epithelial cells caused by buccal cigarette
Drug or health care product in application, which is characterized in that with ultrapure water concussion extract repeatedly, 1 hour every time.
5. smoked plum extractive according to claim 3 is in preparation for preventing and treating the death of mouth epithelial cells caused by buccal cigarette
Drug or health care product in application, which is characterized in that the dry capitulum 100g of dark plum is weighed, after crushing, respectively with 500
The ultrapure water concussion extraction of ml, 300 ml and 200ml are each primary, and each extraction time is 1 hour.
6. smoked plum extractive according to claim 3 is in preparation for preventing and treating the death of mouth epithelial cells caused by buccal cigarette
Drug or health care product in application, which is characterized in that the parameter of noncentricity of high speed freezing centrifuge be 16000 g, 30 minutes.
7. smoked plum extractive according to claim 3 is in preparation for preventing and treating the death of mouth epithelial cells caused by buccal cigarette
Drug or health care product in application, which is characterized in that freeze temperature be -30 DEG C, the time be 24 hours.
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