CN104568785B - A kind of method for determining plant source extractive of volatile oil gaseous phase free radical scavenging capacity - Google Patents
A kind of method for determining plant source extractive of volatile oil gaseous phase free radical scavenging capacity Download PDFInfo
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Abstract
The present invention relates to a kind of method for determining plant source extractive of volatile oil gaseous phase free radical scavenging capacity, step is as follows：1）Take DPPH ethanol solutions to drip in triangular flask bottom, evaporate into it is dry, pipette volatile oil solution be added dropwise gauze on, gauze is hung in triangular flask, it is stoppered bottle stopper and is sealed with preservative film, react certain time, opens tampon, add 2mL absolute ethyl alcohols, shake up, add 18mL absolute ethyl alcohols, shake up, a part is drawn, light absorption value A1 is measured at wavelength 517nm；2）To be not added with the equivalent absolute ethyl alcohol of volatile oil as control, with 1）Middle method is equally operated, and measures light absorption value A2；3）DPPH ethanol solutions are replaced with absolute ethyl alcohol 2mL, with 1）Middle method is equally operated, and measures light absorption value A3；Clearance rate of the volatile oil to DPPH free radicals is calculated by formula.The inventive method is simple to operate, reproducible, and the exploitation for gaseous phase free radical scavenger provides technical foundation.
Description
Technical field
The present invention relates to a kind of method for determining plant source extractive of volatile oil gaseous phase free radical scavenging capacity.
Background technology
The relation such as free radical and aging, inflammation, autoimmune disease and some diseases of old people, endocrine system disease is close
Cut, be particularly it with the relation of tumour by both at home and abroad with the common concern of ambit.British Harman was in 1956
Propose free radical theory：Free radical attack life macromolecule causes tissue cell insult, is the basic original for causing body aging
Cause, is also the important cause of the malignant diseases such as induced tumor.Louisiana, United States stand university and fired it was discovered by researchers that working as
Burn after the particle combination in the free radical and air produced, stable free radical can be generated.This free radical and cigarette coke tar
The free radical of middle discovery is closely similar, and cellular damage can be caused by lung and other tissue resorptions by being inhaled into after human body.It is this thin
Cellular damage can cause asthma, pulmonary emphysema and lung cancer.H.Barry Dellinger point out that people is in the air of medium pollution
The free radical of suction, the free radical that 300 cigarette is sucked was inhaled equivalent to one day.
Epidemiological study has confirmed that the exposure of atmospheric dust can cause the incidence of heart and lung diseases to raise, in toxicity
Learn in research, free radical activity is studied as a key factor of granular biological activity.The free radical relevant with dust
Caused lipid peroxidation injury, genetoxic and cytotoxicity, the emphasis as dust mechanism of causing a disease research in recent years.Environment
Toxicologic study shows that the free radical in urban atmosphere is the potential etiology of the diseases such as angiocarpy, aging, tumour, and freely
The oxidative damage of base is the pathogenic important mechanisms of Atmospheric particulates again.As can be seen here, in atmospheric environment surface particles are stable
Free radical is the threat of health, how to reduce the free-radical contents in air, is the key that human health is safeguarded.
The chemical composition of plant volatile oil is extremely complex, and the volatile oil component of different plants or different parts also has difference
It is different.The chemical composition of plant volatile oil is generally divided into terpenoid, aromatic compound, aliphatic compound and nitrogenous sulfur-bearing
Four kinds of compound.The research report that volatile oil removes free radical is a lot, and the research such as Pan Tianling finds twoflower micromeria herb volatile oil to DPPH
The concentration for removing vigor EC50 of free radical is 0.21%.Sun Wei etc. is according to sample to hexichol for bitter taste diazanyl free radical
(DPPH) scavenging action, evaluates the anti-oxidizing activities of 16 ESSENTIAL OILs.As a result show Radix Angelicae Sinensis, nutmeg, bay,
3 kinds of volatile oil such as tea tree and cloves have stronger anti-oxidizing activities, thyme, Chinese ilex, Pelargonium roseum, lavender and perfume (or spice)
The volatile oil such as cypress have a medium antioxidation, and the antioxidation such as American mint, rosemary, white orchid leaf and You Jiali is not
By force.Amway Corp just had the method that patent protection orange extracted liquor removes free radical in 1998, and Yin Xueqiong etc. is carried
Lemon grass volatile oil is taken and its antioxidation activity is determined with electron spin resonance and found, mass fraction is 1% perfume (or spice)
Thatch volatile oil ethanol solution is about 32% to the clearance rate of hydroxyl radical free radical.Zhao Xin etc. report the volatility of sweet orange volatile oil into
Divide predominantly limonene, be secondly nopinene, β-phellandrene, linalool, australene etc., show through DPPH radicals scavengings experiment
68.07% volatile oil is 50% to free radical scavenging activity.
The content of the invention
It is an object of the invention to provide a kind of method for determining plant source extractive of volatile oil gaseous phase free radical scavenging capacity,
This method is simple to operate, reproducible, and the exploitation for gaseous phase free radical scavenger provides technical foundation.
In order to realize above-mentioned purpose, present invention employs following technical scheme：
A kind of method for determining plant source extractive of volatile oil gaseous phase free radical scavenging capacity, this method includes following step
Suddenly：
1) take DPPH ethanol solution 2-3mL drops in 100mL triangular flasks bottom, room temperature volatilizees absolute ethyl alcohol to dry, pipettes
Volatile oil solution is added dropwise on gauze, and gauze is hung in triangular flask rapidly, is stoppered triangle bottle stopper, uses preservative film sealing bottle cork, instead
Answer certain time, open tampon, add 2mL absolute ethyl alcohols in triangular flask, fully shake up, add 18mL absolute ethyl alcohols, shake
It is even, a part is drawn, light absorption value A1 is measured at wavelength 517nm；
2) to be not added with the equivalent absolute ethyl alcohol of volatile oil as control, equally operated in method in 1), measure light absorption value A2；
3) DPPH ethanol solutions are replaced with absolute ethyl alcohol 2mL, is equally operated in method in 1), measure light absorption value A3；
Clearance rate of the volatile oil to DPPH free radicals is calculated as follows:
Preferably, described DPPH ethanol solution concentrations are 25 μ g/mL.
The present invention is as a result of above-mentioned technical scheme, and this method is simple to operate, reproducible, is that gaseous phase free radical is clear
Except the exploitation of agent provides technical foundation.
Embodiment
Illustrate technical scheme, but protection scope of the present invention not limited to this with specific embodiment below：
Embodiment 1：Plant volatile oil extraction and its to the Scavenging activity of gas phase system DPPH free radicals.
1st, the extraction of plant volatile oil
Using steam distillation, raw material is 1 with water weight proportion：4-5, is 150-170 DEG C in Extracting temperature, during extraction
Between 5-7h, steam imports the liquid mixture that water and volatile oil are obtained in condenser through pot top gooseneck in heating process, by oil
Volatile oil product is can obtain after water separation.Its result such as following table
The volatile oil yield of table 1
| Volatile oil kind | Materials (g) | It must measure (ml) | Yield (%) |
| Tea tree flowers volatile oil | 500 | 1.0 | 0.2 |
| Red bayberry leaf volatile oil | 500 | 0.2 | 0.04 |
| Lavender | 500 | 1.2 | 0.24 |
| Fructus Citri Sarcodactylis volatile oil | 500 | 0.4 | 0.08 |
| Folium artemisiae argyi | 500 | 0.9 | 0.18 |
| Cloves | 500 | 1.3 | 0.26 |
| Lemon volatile oil | 500 | 2.3 | 0.46 |
| Sweet orange peel | 500 | 5.6 | 1.12 |
| Pomelo peel volatile oil | 500 | 4.1 | 0.82 |
| Pericarpium Citri tangerinae volatile oil | 500 | 5.2 | 1.04 |
Volatile oil originates from fragrant plant (Aromatic Herbst), the face cream glands of different plants be distributed in petal, leaf,
On fruit skin, rhizome or trunk, sachet is refined after extraction, volatile oil is become, therefore volatile oil yield is generally relatively low, it is fresh
Sweet orange peel volatile oil content highest, extraction by steam distillation yield is up to 1.12%, and myrica rubra leaf volatile oil content is minimum, yield
Only 0.04%.
2nd, the radicals scavenging of plant volatile oil gas phase system is tested
1. DPPH ethanol solutions (concentration is 25 μ g/mL) 2-3mL drops are taken in 100mL triangular flasks bottom, room temperature volatilization nothing
Water-ethanol is pipetted on the μ L of the volatile oil solution 50 dropwise addition gauzes of various concentrations, gauze is hung in triangular flask rapidly, is stoppered to dry
Triangle bottle stopper, uses preservative film sealing bottle cork, reacts certain time, opens bottle stopper, adds 2mL absolute ethyl alcohols in triangular flask, fills
Divide and shake up, add 18mL absolute ethyl alcohols, shake up, draw a part, light absorption value A1 is measured at wavelength 517nm；
2. to be not added with the equivalent absolute ethyl alcohol of volatile oil as control, equally operated in method in (1), measure light absorption value A2；
3. DPPH ethanol solutions are replaced with absolute ethyl alcohol 2mL, is equally operated in method in (1), measure light absorption value A3；Press
Following formula calculates clearance rate of the volatile oil to DPPH free radicals:
Folium artemisiae argyi, cloves, lavender, sweet orange peel, orange peel, pomelo peel, lemon peel, bergamot leaves, red bayberry are obtained by the above process
Leaf, Tea Flower etc. more than 10 plant plant material extract obtained volatile oil its remove DPPH free radicals ability it is as follows：
According to experimental result, orange peel volatile oil is y to the equation of linear regression of the removing of DPPH free radicals in gas phase system
=203.68x+41.531, coefficient R 2=0.984, under 0.01 μ g/mL-0.0125 μ g/mL volatile oil concentration range
With preferable linear dependence, half clean-up effect concentration EC50 can be obtained for 0.0416 μ g/mL by linear equation.
According to experimental result, shaddock volatile oil is y to the equation of linear regression of the removing of DPPH free radicals in gas phase system
=351.72x+46.835, coefficient R 2=0.8647, under 0.01 μ g/mL-0.0125 μ g/mL volatile oil concentration range
With preferable linear dependence, half clean-up effect concentration EC50 can be obtained for 0.009 μ g/mL by linear equation.
According to experimental result, linear regression side of the lavender orange peel volatile oil to the removing of DPPH free radicals in gas phase system
Journey is y=278.19x+40.006, coefficient R 2=0.9201, in 0.01 μ g/mL-0.0125 μ g/mL volatilization oil concentration
There is preferable linear dependence under scope, half clean-up effect concentration EC50 can be obtained for 0.036 μ g/ by linear equation
mL。
According to experimental result, sweet orange peel volatile oil is to the equation of linear regression of the removing of DPPH free radicals in gas phase system
Y=224.22x+38.904, coefficient R 2=0.9875, in 0.01 μ g/mL-0.0125 μ g/mL volatile oil concentration range
There is down preferable linear dependence, half clean-up effect concentration EC50 can be obtained for 0.049 μ g/mL by linear equation.
According to experimental result, Buddha's hand glycosides volatile oil is to the equation of linear regression of the removing of DPPH free radicals in gas phase system
Y=426.56x+37.429, coefficient R 2=0.9432, in 0.01 μ g/mL-0.0125 μ g/mL volatile oil concentration range
There is down preferable linear dependence, half clean-up effect concentration EC50 can be obtained for 0.03 μ g/mL by linear equation.
According to experimental result, lemon peel volatile oil is to the equation of linear regression of the removing of DPPH free radicals in gas phase system
Y=206.22x+44.977, coefficient R 2=0.8716, in 0.01 μ g/mL-0.0125 μ g/mL volatile oil concentration range
There is down preferable linear dependence, half clean-up effect concentration EC50 can be obtained for 0.024 μ g/mL by linear equation.
According to experimental result, tea tree flowers volatile oil is to the equation of linear regression of the removing of DPPH free radicals in gas phase system
Y=402.87x+41.339, coefficient R 2=0.9442, in 0.01 μ g/mL-0.0125 μ g/mL volatile oil concentration range
There is down preferable linear dependence, half clean-up effect concentration EC50 can be obtained for 0.02 μ g/mL by linear equation.
According to experimental result, volatile clove oil is y to the equation of linear regression of the removing of DPPH free radicals in gas phase system
=464.28x+20.282, coefficient R 2=0.9291, under 0.01 μ g/mL-0.0125 μ g/mL volatile oil concentration range
With preferable linear dependence, half clean-up effect concentration EC50 can be obtained for 0.06 μ g/mL by linear equation.
According to experimental result, volatile argyi leaf oil is y to the equation of linear regression of the removing of DPPH free radicals in gas phase system
=156.39x+44.767, coefficient R 2=0.8604, under 0.01 μ g/mL-0.0125 μ g/mL volatile oil concentration range
With preferable linear dependence, half clean-up effect concentration EC50 can be obtained for 0.03 μ g/mL by linear equation.
According to experimental result, red bayberry leaf volatile oil is to the equation of linear regression of the removing of DPPH free radicals in gas phase system
Y=603.52x+17.335, coefficient R 2=0.9558, in 0.01 μ g/mL-0.0125 μ g/mL volatile oil concentration range
There is down preferable linear dependence, half clean-up effect concentration EC50 can be obtained for 0.05 μ g/mL by linear equation.
Embodiment 2：The radicals scavenging synergism experiment of compounding two-by-two of different plant volatile oils.
According to the EC50 (free radical half clean-up effect concentration) of the different plant volatile oils calculated in embodiment 1, press
The yield for extracting volatile oil according to different plant materials is converted into material content, with 1：1 concentration ratio intersects compounding two-by-two, is extracting
Under 150-170 DEG C of temperature, extraction time 5-7h, volatile oil is obtained by water-oil separating.It is clear through DPPH free radicals in gas phase system
Except experiment, concrete operations are 1. to take DPPH ethanol solutions (concentration is 25 μ g/mL) 2-3mL drops in 100mL triangular flasks bottom, room
Temperature volatilization absolute ethyl alcohol is pipetted on the μ L of the volatile oil solution 50 dropwise addition gauzes of various concentrations, gauze hangs on rapidly triangle to dry
In bottle, triangle bottle stopper is stoppered, preservative film sealing bottle cork is used, certain time is reacted, bottle stopper is opened, 2mL absolute ethyl alcohols are added in three
In the bottle of angle, fully shake up, add 18mL absolute ethyl alcohols, shake up, draw a part, light absorption value A1 is measured at wavelength 517nm；
2. to be not added with the equivalent absolute ethyl alcohol of volatile oil as control, equally operated in method in (1), measure light absorption value A2；
3. DPPH ethanol solutions are replaced with absolute ethyl alcohol 2mL, is equally operated in method in (1), measure light absorption value A3；Press
Following formula calculates clearance rate of the volatile oil to DPPH free radicals:
According to above method, result is obtained as follows：
From experimental result, gaseous phase free radical clearance rate is obviously improved after compounding two-by-two, its clearance rate exceedes
60% clearance rate has a 1+6 (bergamot volatile oil+lemon peel volatile oil), 2+5 (red bayberry leaf volatile oil+Pericarpium Citri tangerinae volatile oil), 3
+ 5 (pomelo peel volatile oil+Pericarpium Citri tangerinae volatile oil), 3+9 (pomelo peel volatile oil+lavenderessential oil), 5+9 (Pericarpium Citri tangerinae volatile oils
+ lavenderessential oil), 7+8 (volatile argyi leaf oil+sweet orange peel volatile oil), 7+9 (volatile argyi leaf oil+lavenderessential oil) exceed
70% clearance rate has 5+9 (Pericarpium Citri tangerinae volatile oil+lavenderessential oil), a 7+9 (volatile argyi leaf oil+lavenderessential oil), its
The synergistic effect of middle 2+3 (red bayberry volatile oil+pomelo peel volatile oil) is the most notable, has exceeded 80% clearance rate, therefore compounding
Middle selection red bayberry leaf volatile oil, pomelo peel volatile oil, in addition Pericarpium Citri tangerinae volatile oil and lavenderessential oil and other volatile oil
Compounding also accordingly improves the clearance rate to free radical, but red bayberry leaf volatile oil is looked into lavenderessential oil compounding effect, and its is clear
Except rate is reduced to 20%, with obvious antagonism.Therefore experiment is using red bayberry leaf volatile oil, pomelo peel volatile oil and orange peel
Three kinds of volatile oil, which carries out mixing compounding, should be able to obtain preferably removing free radical effect.
Embodiment 3：The extraction of the Mixed chaotic sequences of myrica rubra leaf, pomelo peel and orange peel and its free radical scavenging activity.
According to the synergism experiment result of embodiment 2, myrica rubra leaf, three kinds of materials of pomelo peel and orange peel are chosen, according to red bayberry phyllogen
25-50 parts of the parts by weight (in terms of weight in wet base) of material, 2-7 parts of the parts by weight (in terms of weight in wet base) of pomelo peel, the parts by weight of orange peel
Number is mixed, with 1 10-20 parts (in terms of weight in wet base) after crushing：4-5 material-water ratio, in 150-170 DEG C of Extracting temperature, extraction time 5-
Under 7h, volatile oil is obtained by water-oil separating.Tested through DPPH radicals scavengings in gas phase system, concrete operations are 1. to take
DPPH ethanol solutions (concentration is 25 μ g/mL) 2-3mL drops are in 100mL triangular flasks bottom, and room temperature volatilizees absolute ethyl alcohol to dry, moves
The μ L of volatile oil solution 50 of various concentrations are taken to be added dropwise on gauze, gauze is hung in triangular flask rapidly, is stoppered triangle bottle stopper, with guarantor
Fresh film sealing bottle cork, reacts certain time, opens bottle stopper, adds 2mL absolute ethyl alcohols in triangular flask, fully shakes up, add
18mL absolute ethyl alcohols, shake up, and draw a part, light absorption value A1 is measured at wavelength 517nm；
2. to be not added with the equivalent absolute ethyl alcohol of volatile oil as control, equally operated in method in (1), measure light absorption value A2；
3. DPPH ethanol solutions are replaced with absolute ethyl alcohol 2mL, is equally operated in method in (1), measure light absorption value A3；Press
Following formula calculates clearance rate of the volatile oil to DPPH free radicals:
The clearance rate that gas phase system DPPH free radicals are measured with above method is 89-93%.
Removing of the volatile oil mixing compounding that orange peel, three kinds of raw materials of pomelo peel and myrica rubra leaf are extracted to DPPH free radicals is equal
Greatly improve, with stronger gaseous phase free radical scavenger DEVELOPMENT PROSPECT.
Above-described embodiment is the preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes made without departing from the present invention should be equivalent substitute mode, be included in the guarantor of the present invention
Within the scope of shield.
Claims (1)
1. a kind of method for determining plant source extractive of volatile oil gaseous phase free radical scavenging capacity, it is characterised in that this method includes
Following step：
1）Take DPPH ethanol solution 2-3mL drops in 100mL triangular flasks bottom, DPPH ethanol solution concentrations are 25 μ g/mL,
Room temperature volatilizees absolute ethyl alcohol to dry, pipettes volatile oil solution and is added dropwise on gauze, gauze is hung in triangular flask rapidly, is stoppered triangle
Bottle stopper, uses preservative film sealing bottle cork, reacts certain time, opens tampon, adds 2mL absolute ethyl alcohols in triangular flask, fully shakes
It is even, 18mL absolute ethyl alcohols are added, are shaken up, a part is drawn, light absorption value A1 is measured at wavelength 517nm；Described volatile oil
Solution uses steam distillation, and raw material is 1 with water weight proportion：4-5, is 150-170 DEG C, extraction time 5- in Extracting temperature
Steam imports the liquid mixture that water and volatile oil are obtained in condenser through pot top gooseneck in 7h, heating process, by profit point
Volatile oil solution is can obtain from rear；Described volatile oil solution be folium artemisiae argyi, cloves, lavender, sweet orange peel, orange peel, pomelo peel,
Lemon peel, bergamot leaves, myrica rubra leaf, Tea Flower raw material extract obtained volatile oil；
2）To be not added with the equivalent absolute ethyl alcohol of volatile oil as control, with 1）Middle method is equally operated, and measures light absorption value A2；
3）DPPH ethanol solutions are replaced with absolute ethyl alcohol 2mL, with 1）Middle method is equally operated, and measures light absorption value A3；
Clearance rate of the volatile oil to DPPH free radicals is calculated as follows:
Clearance rate=×100%；
4）The ability for calculating volatile oil solution removing DPPH free radicals is as follows：
Orange peel volatile oil is y=203.68x+41.531, phase to the equation of linear regression of the removing of DPPH free radicals in gas phase system
Relation number R2=0.984, has preferable linear correlation under 0.01 μ g/mL-0.0125 μ g/mL volatile oil concentration range
Property, half clean-up effect concentration EC50 can be obtained for 0.0416 μ g/mL by linear equation；
Shaddock volatile oil is y=351.72x+46.835, phase to the equation of linear regression of the removing of DPPH free radicals in gas phase system
Relation number R2=0.8647, has preferable linear correlation under 0.01 μ g/mL-0.0125 μ g/mL volatile oil concentration range
Property, half clean-up effect concentration EC50 can be obtained for 0.009 μ g/mL by linear equation；
Lavender orange peel volatile oil is y=278.19x+ to the equation of linear regression of the removing of DPPH free radicals in gas phase system
40.006, coefficient R 2=0.9201 has preferable under 0.01 μ g/mL-0.0125 μ g/mL volatile oil concentration range
Linear dependence, half clean-up effect concentration EC50 can be obtained for 0.036 μ g/mL by linear equation；
Sweet orange peel volatile oil is y=224.22x+38.904 to the equation of linear regression of the removing of DPPH free radicals in gas phase system,
Coefficient R 2=0.9875, has preferably linear phase under 0.01 μ g/mL-0.0125 μ g/mL volatile oil concentration range
Guan Xing, half clean-up effect concentration EC50 can be obtained for 0.049 μ g/mL by linear equation；
Buddha's hand glycosides volatile oil is y=426.56x+37.429 to the equation of linear regression of the removing of DPPH free radicals in gas phase system,
Coefficient R 2=0.9432, has preferably linear phase under 0.01 μ g/mL-0.0125 μ g/mL volatile oil concentration range
Guan Xing, half clean-up effect concentration EC50 can be obtained for 0.03 μ g/mL by linear equation；
Lemon peel volatile oil is y=206.22x+44.977 to the equation of linear regression of the removing of DPPH free radicals in gas phase system,
Coefficient R 2=0.8716, has preferably linear phase under 0.01 μ g/mL-0.0125 μ g/mL volatile oil concentration range
Guan Xing, half clean-up effect concentration EC50 can be obtained for 0.024 μ g/mL by linear equation；
Tea tree flowers volatile oil is y=402.87x+41.339 to the equation of linear regression of the removing of DPPH free radicals in gas phase system,
Coefficient R 2=0.9442, has preferably linear phase under 0.01 μ g/mL-0.0125 μ g/mL volatile oil concentration range
Guan Xing, half clean-up effect concentration EC50 can be obtained for 0.02 μ g/mL by linear equation；
Volatile clove oil is y=464.28x+20.282, phase to the equation of linear regression of the removing of DPPH free radicals in gas phase system
Relation number R2=0.9291, has preferable linear correlation under 0.01 μ g/mL-0.0125 μ g/mL volatile oil concentration range
Property, half clean-up effect concentration EC50 can be obtained for 0.06 μ g/mL by linear equation；
Volatile argyi leaf oil is y=156.39x+44.767, phase to the equation of linear regression of the removing of DPPH free radicals in gas phase system
Relation number R2=0.8604, has preferable linear correlation under 0.01 μ g/mL-0.0125 μ g/mL volatile oil concentration range
Property, half clean-up effect concentration EC50 can be obtained for 0.03 μ g/mL by linear equation；
Red bayberry leaf volatile oil is y=603.52x+17.335 to the equation of linear regression of the removing of DPPH free radicals in gas phase system,
Coefficient R 2=0.9558, has preferably linear phase under 0.01 μ g/mL-0.0125 μ g/mL volatile oil concentration range
Guan Xing, half clean-up effect concentration EC50 can be obtained for 0.05 μ g/mL by linear equation.
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| Use of different methods for testing antioxidative activity of oregano essential oil;T. Kulisic等;《Food Chemistry》;20030724;第635页 * |
| 功能性苦荞酒的研制;李云龙等;《中国杂粮研究》;20071101;第92-293页 * |
| 杨梅叶水溶性提取物的抗氧化、抗菌活性与防腐应用;杨君等;《中国食品添加剂开发应用》;20140228;全文 * |
| 用清除有机自由基DPPH法评价植物抗氧化能力;彭长连等;《生物化学与生物物理进展》;20000430;第27卷(第6期);全文 * |
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