CN114480017A - Preparation method of component tobacco seed oil with antioxidant activity, antioxidant activity evaluation method and application - Google Patents
Preparation method of component tobacco seed oil with antioxidant activity, antioxidant activity evaluation method and application Download PDFInfo
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- CN114480017A CN114480017A CN202210173520.7A CN202210173520A CN114480017A CN 114480017 A CN114480017 A CN 114480017A CN 202210173520 A CN202210173520 A CN 202210173520A CN 114480017 A CN114480017 A CN 114480017A
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- seed oil
- tobacco seed
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B3/00—Refining fats or fatty oils
- C11B3/001—Refining fats or fatty oils by a combination of two or more of the means hereafter
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
- A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
- A23D9/007—Other edible oils or fats, e.g. shortenings, cooking oils characterised by ingredients other than fatty acid triglycerides
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
- A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
- A23D9/02—Other edible oils or fats, e.g. shortenings, cooking oils characterised by the production or working-up
- A23D9/04—Working-up
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
- C11B1/10—Production of fats or fatty oils from raw materials by extracting
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B3/00—Refining fats or fatty oils
- C11B3/003—Refining fats or fatty oils by enzymes or microorganisms, living or dead
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B3/00—Refining fats or fatty oils
- C11B3/10—Refining fats or fatty oils by adsorption
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B3/00—Refining fats or fatty oils
- C11B3/12—Refining fats or fatty oils by distillation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- Oil, Petroleum & Natural Gas (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Botany (AREA)
- Nutrition Science (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Mycology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Cosmetics (AREA)
- Fats And Perfumes (AREA)
Abstract
The invention discloses a preparation method of a component tobacco seed oil with antioxidant activity, an antioxidant activity evaluation method and application. The preparation method of the tobacco seed oil with the antioxidant active ingredient comprises the steps of selecting raw materials, and extracting the raw materials by a Soxhlet extraction method to obtain tobacco seed primary oil; adding a biological adsorbent into the tobacco seed primary oil, and placing the tobacco seed primary oil in a constant-temperature shaking table for oscillation adsorption reaction for 6-8 hours; after the reaction is finished, separating and filtering the mixture by using a microporous filter membrane to remove the biological adsorbent to obtain a semi-finished product of the tobacco seed oil; and (3) putting the semi-finished product of the tobacco seed oil into a molecular distillation instrument for carrying out three times of molecular distillation, and combining three fractions of the tobacco seed oil subjected to the three times of molecular distillation to obtain the tobacco seed oil with high purity, edible and antioxidant active ingredients. The tobacco seed oil is added into edible oil or health care products after the evaluation of the antioxidant activity is qualified, so that the antioxidant performance of the tobacco seed oil is exerted.
Description
Technical Field
The invention belongs to the technical field of tobacco products, and particularly relates to a preparation method of tobacco seed oil with an antioxidant active ingredient, an antioxidant activity evaluation method and application thereof.
Background
Tobacco seeds are seeds of tobacco, and the application of tobacco seeds mainly focuses on variety breeding and industrial and commercial raw material production for a long time, so research work aiming at tobacco seeds mainly focuses on improving the yield and quality of the seeds. But the research work related to the aspects of multifunctional exploration of tobacco seeds, development prospects of raw materials and products, market application range and the like is very rare, and the technical gap is obvious. Meanwhile, in field production, the tobacco seed propagation coefficient is high, the tobacco seed yield far exceeds the raw material requirement of industrial and commercial tobacco leaf production, and the surplus tobacco seed quantity is large. The technical insufficiency and the stock surplus limit the utilization of tobacco seed resources to a great extent. In recent years, many reports have been made on the utilization of resources such as waste tobacco leaves and tobacco stems, but few reports have been made on the comprehensive utilization of tobacco seeds.
The tobacco seeds are rich in fatty oil, are good fatty oil raw materials, have the oil content of 40-50 percent, and have multiple purposes in the oil chemical industry, such as being used for preparing soap products in the soap manufacturing industry and being used as raw materials for preparing alkyd resin paint in the paint manufacturing industry, but the tobacco seed oil extracted by the Soxhlet extraction method cannot be directly eaten. The tobacco seed as one of tobacco products has great scientific research and application potential, so that the development of a tobacco seed oil and the exploration of the functional application of the tobacco seed oil are very important.
In view of the above, it is necessary to research a method for preparing a tobacco seed oil with an antioxidant activity, a method for evaluating antioxidant activity, and applications thereof for solving the above technical problems.
Disclosure of Invention
The first purpose of the invention is to provide a preparation method of the tobacco seed oil with the antioxidant active ingredients, the second purpose of the invention is to provide an evaluation method of the antioxidant activity of the tobacco seed oil, and the third purpose of the invention is to provide application of the tobacco seed oil with the antioxidant active ingredients.
The first purpose of the invention is realized by a method for preparing the tobacco seed oil with the antioxidant active ingredient, which comprises the following steps:
(1) selecting raw materials: collecting mature tobacco capsule, and air-drying until the tobacco capsule is dry; threshing tobacco capsules, winnowing to obtain plump tobacco seeds, cleaning the plump tobacco seeds, and drying until the moisture content is reduced to 8% -10% for later use;
(2) extracting the plump tobacco seeds dried in the step (1) by using a Soxhlet extraction method to obtain tobacco seed primary oil;
(3) adding a biological adsorbent into the tobacco seed primary oil obtained in the step (2), and placing the mixture in a constant-temperature shaking table for oscillation adsorption reaction for 6-8 hours;
(4) after the reaction in the step (3) is finished, separating and filtering the mixture by using a microporous filter membrane to remove the biological adsorbent to obtain a semi-finished product of the tobacco seed oil;
(5) putting the semi-finished product of the tobacco seed oil in the step (4) into a molecular distillation instrument, carrying out first molecular distillation at 79-81 ℃ under the condition of 100Pa, and obtaining tobacco seed oil L1 with first fraction as a first-stage light component after condensation; continuing to perform secondary molecular distillation on the first distillation residue at 99-101 ℃ under the condition of 10Pa, and condensing to obtain second fraction which is smoke seed oil L2 of a second-stage light component; continuously carrying out three-stage molecular distillation on the second distillation residue at the temperature of 119-121 ℃ and under the condition of 1Pa, and condensing to obtain smoke seed oil L3 with a third fraction as a three-stage light component;
(6) and (4) combining the tobacco seed oil L1, the tobacco seed oil L2 and the tobacco seed oil L3 in the step (5) to obtain the tobacco seed oil with the antioxidant active ingredients.
Preferably, the Soxhlet extraction method in the step (2) is that dried plump tobacco seeds are ground and extracted for 8-9h at 84-86 ℃ by using n-hexane as a solvent according to the material-liquid ratio of 1: 18.
Preferably, the biosorbent in step (3) is yeast powder, and the mass ratio of the yeast powder to the primary tobacco seed oil is 1: 20.
Preferably, the pore size of the microporous filter membrane in the step (4) is 0.32 μm.
Preferably, the feeding speed of the molecular distillation apparatus in the step (5) is 50mL/h, and the rotating speed of a rotor is 120 r/min.
The second purpose of the invention is realized by a method for evaluating the antioxidant activity of the tobacco seed oil, which comprises the following steps:
s1: performing GC-MS detection on the tobacco seed oil with the antioxidant active ingredients according to any one of claims 1 to 5 to detect the content of fatty acid and the content of volatile ingredients in the tobacco seed oil with the antioxidant active ingredients;
s2: measuring the in vitro antioxidant activity and the in vivo cell level antioxidant activity of Vc by taking Vc as a positive control to obtain an in vitro antioxidant activity value and an in vivo cell level antioxidant activity value of Vc;
s3: measuring the in vitro antioxidant activity and the in vivo cell level antioxidant activity of the tobacco seed oil to obtain an in vitro antioxidant activity value and an in vivo cell level antioxidant activity value of the tobacco seed oil;
s4: comparing the Vc in-vitro antioxidant activity value in the step S2 with the in-vitro antioxidant activity value of the tobacco seed oil in the step S3 to obtain the in-vitro antioxidant activity capacity of the tobacco seed oil;
s5: comparing the Vc in-vivo cell level antioxidant activity value in the step S2 with the in-vivo cell level antioxidant activity value of the tobacco seed oil in the step S3 to obtain the in-vivo antioxidant activity capacity of the tobacco seed oil;
s6: and (4) evaluating the antioxidant activity of the tobacco seed oil by combining the in-vitro antioxidant activity of the tobacco seed oil in the step S4, the in-vivo antioxidant activity of the tobacco seed oil in the step S5, the fatty acid content and the volatile component content of the tobacco seed oil in the step S1.
Preferably, the GC-MS detection conditions of the fatty acid content in step S1 are:
GC detection conditions are as follows: the temperature of a sample inlet is 240 ℃; the carrier gas is 99.999 percent high-purity helium, the flow rate is 1mL/min, and split-flow sample injection is not carried out; chromatographic column model HP-5 quartz capillary column of 30m × 0.32mm × 0.25 μm, temperature programmed conditions: the initial temperature of the column oven is 40 ℃, and then the temperature is increased to 80 ℃ at the speed of 1 ℃/min; then increasing the temperature to 250 ℃ at the speed of 20 ℃/min, and keeping the temperature for 10 min;
MS detection conditions: the ion source is an EI source, the electron energy is 70eV, the interface temperature is 250 ℃, the ion source temperature is 230 ℃, the mass scanning range is 30-540m/z, the solvent delay is 3min, and the full scanning mode is adopted.
Preferably, the determination method of the in vitro antioxidant activity value of Vc in the step S2 and the in vitro antioxidant activity value of the tobacco seed oil in the step S3 are both: the comprehensive measurement is carried out by using two or more of DPPH free radical scavenging ability, ABTS free radical scavenging ability, hydroxyl free radical scavenging ability and FRAP reducing ability.
Preferably, the determination method of the in vivo antioxidant activity value of Vc in the step S2 and the determination method of the in vivo antioxidant activity value of the tobacco seed oil in the step S3 are both as follows: h pair by using tobacco seed oil2O2Inducing active oxygen content in HepG2 cells and tobacco seed oil pair H2O2Inducing HepG2 cell apoptosis value and tobacco seed oil pair H2O2And comprehensively measuring the content of GSH, SOD and CAT enzymes or the content of GSH and CAT enzymes in the induced HepG2 cells.
Preferably, the tobacco seed oil with the antioxidant active ingredients is added into edible oil or health care products after the antioxidant activity evaluation is qualified.
The third purpose of the invention is realized by the application of the tobacco seed oil with the antioxidant active ingredient, and the tobacco seed oil with the antioxidant active ingredient is added into edible oil or health care products after the antioxidant activity is qualified.
Compared with the prior art, the invention has the following technical effects:
1. the invention extracts the tobacco seed primary oil from the tobacco seeds by a Soxhlet extraction method, and then carries out three times of molecular distillation on the semi-finished product of the tobacco seed oil which is adsorbed by a biological adsorbent and separated and filtered by a microporous membrane to enrich the effective active ingredients of the semi-finished product, thereby obtaining the edible functional tobacco seed oil with antioxidant active ingredients.
2. According to the invention, the yeast powder is adopted as a biological adsorbent for adsorption, and the primary tobacco seed oil is separated and filtered by adopting the microporous filter membrane, so that the solvent used in primary tobacco seed oil extraction, heavy metals, salts, pigments, impurities and the like in the tobacco seed oil can be effectively removed.
3. According to the invention, the semi-finished product of the tobacco seed oil is subjected to three times of molecular distillation at different temperatures and pressures, so that the antioxidant active ingredients of the tobacco seed oil are effectively retained, the impurity ingredients in the tobacco seed oil can be separated again, and the high-purity edible functional tobacco seed oil with the antioxidant active ingredients is obtained through three times of molecular distillation.
4. The invention comprehensively evaluates the antioxidant activity of the tobacco seed oil to determine that the tobacco seed oil has antioxidant active ingredients; the tobacco seed oil qualified by antioxidant activity evaluation is added into edible oil or health care products to exert the antioxidant performance of the tobacco seed oil.
5. The invention not only improves the utilization rate of tobacco resources, but also widens the application and market application prospect of the tobacco seed oil.
Drawings
FIG. 1 is a graph of Reactive Oxygen Species (ROS) production inhibition by cured tobacco NC89 seed oil in example 1 of the present invention; (wherein: K: blank control group; Y: positive control group; M: model group;#P<0.05vs group K;*P<0.05vs group M);
FIG. 2 shows the cured tobacco NC89 tobacco seed oil pair H in example 1 of the present invention2O2Graph of induction of HepG2 apoptosis (a) and rate of apoptosis (B);
FIG. 3 shows cured tobacco according to example 1 of the present inventionNC89 tobacco seed oil Pair H2O2Graph of effect on induction of GSH, SOD and CAT enzyme activity in HepG2 cells; in FIG. 3, A is a graph showing the change in GSH enzyme activity, B is a graph showing the change in SOD enzyme activity, and C is a graph showing the change in CAT enzyme activity;
FIG. 4 is a graph of the inhibition of reactive oxygen species ROS generation by the aromatic tobacco Baoshan No. 4 tobacco seed oil in example 2 of the present invention; (wherein: K: blank control group; Y: positive control group; M: model group;#P<0.05vs group K;*P<0.05vs group M);
FIG. 5 shows the aromatic tobacco Baoshan No. 4 tobacco seed oil pair H in example 2 of the present invention2O2Graph of induction of HepG2 apoptosis (a) and rate of apoptosis (B);
FIG. 6 shows aromatic tobacco Baoshan No. 4 tobacco seed oil pair H in example 2 of the present invention2O2Graph of effect on induction of GSH, SOD and CAT enzyme activity in HepG2 cells; in FIG. 6, A is a graph showing the change in GSH enzyme activity, B is a graph showing the change in SOD enzyme activity, and C is a graph showing the change in CAT enzyme activity;
FIG. 7 is a graph showing that wild tobacco Rustica seed oil inhibits reactive oxygen species ROS generation in example 3 of the present invention; (wherein: K: blank control group; Y: positive control group; M: model group;#P<0.05vs group K;*P<0.05vs group M);
FIG. 8 shows the wild tobacco Rustica tobacco seed oil pair H in example 3 of the present invention2O2Graph of induction of HepG2 apoptosis (a) and rate of apoptosis (B);
FIG. 9 shows the wild tobacco Rustica tobacco seed oil pair H in example 3 of the present invention2O2Graphs of the effect of inducing GSH and CAT enzyme activity in HepG2 cells; in FIG. 9, A is a graph showing a change in GSH enzyme activity and B is a graph showing a change in CAT enzyme activity;
FIG. 10 is a graph of reactive oxygen species ROS production inhibition by cigar Yunyun No. 2 seed oil in example 4 of the present invention; (wherein: K: blank control group; Y: positive control group; M: model group;#P<0.05vs group K;*P<0.05vs group M);
FIG. 11 shows the cigar Yunye No. 2 tobacco seed oil pair H in example 4 of the present invention2O2Induction of HepG2 apoptosis (A) and apoptosis Rate (B)A drawing;
FIG. 12 shows the cigar Yunye No. 2 tobacco seed oil pair H in example 4 of the present invention2O2Graph of effect on induction of GSH, SOD and CAT enzyme activity in HepG2 cells;
in FIG. 12, A is a graph showing the change in GSH enzyme activity, B is a graph showing the change in SOD enzyme activity, and C is a graph showing the change in CAT enzyme activity.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to be limiting in any way, and any modifications or alterations based on the teachings of the present invention are intended to fall within the scope of the present invention.
Example 1
The embodiment takes the NC89 variety in the flue-cured tobacco as an example.
1.1A preparation method of NC89 tobacco seed oil with antioxidant active ingredients comprises the following steps:
(1) selecting raw materials: collecting mature NC89 tobacco capsule, and air-drying to NC89 tobacco capsule; threshing tobacco capsules, winnowing to obtain plump NC89 tobacco seeds, cleaning the plump tobacco seeds, and drying until the moisture content is reduced to 9% for later use;
(2) grinding the dried full NC89 tobacco seeds, and leaching for 9 hours at 85 ℃ by using n-hexane as a solvent according to a material-liquid ratio of 1:18 to obtain NC89 tobacco seed primary oil;
(3) adding yeast powder biological adsorbent into NC89 tobacco seed primary oil, placing in a constant temperature shaking table, and performing oscillation adsorption reaction for 8 h; the mass ratio of the yeast powder to the tobacco seed primary oil is 1: 20;
(4) after the reaction is finished, separating and filtering by using a 0.32 mu m microporous filter membrane to remove yeast powder to obtain a semi-finished product of NC89 tobacco seed oil;
(5) placing the NC89 tobacco seed oil semi-finished product into a molecular distillation apparatus, wherein the feeding speed of the molecular distillation apparatus is 50mL/h, and the rotating speed of a rotor is 120 r/min; performing first molecular distillation at 80 deg.C under 100Pa to obtain first fraction of oleum Nicotianae L1 as first-stage light component; continuing to perform secondary molecular distillation on the primary distillation residues at 100 ℃ under the condition of 10Pa, and condensing to obtain a second fraction which is the smoke seed oil L2 of a second-stage light component; continuously carrying out three-stage molecular distillation on the second distillation residue at the temperature of 120 ℃ and under the condition of 1Pa, and condensing to obtain tobacco seed oil L3 with a third fraction as a third-stage light component;
(6) and (3) combining the tobacco seed oil L1, the tobacco seed oil L2 and the tobacco seed oil L3 to obtain the NC89 tobacco seed oil with antioxidant active ingredients.
1.2, carrying out GC-MS detection on NC89 tobacco seed oil with antioxidant active ingredients to determine the content of fatty acid and the content of volatile ingredients in the tobacco seed oil.
1.2.1 determination method of fatty acid content: and (3) extracting and enriching NC89 tobacco seed oil samples by adopting a headspace solid phase microextraction method (HS-SPME), wherein the model of the extraction fiber head is 50/30 mu m DVB/CAR/PDMS. Before each use of the extraction fiber, the extraction fiber needs to be aged for 3min at a GC-MS injection port of 240 ℃. Accurately weighing 3mL NC89 tobacco seed oil with antioxidant active ingredient, adding into 20mL headspace bottle, immediately sealing, and extracting at 40 deg.C for 20 min. After extraction, the solid phase micro-extraction fiber needle is taken out, and then inserted into a GC injection port for desorption (3min, 240 ℃) and subsequent GC-MS analysis.
Specifically, the GC detection conditions were: the temperature of a sample inlet is 240 ℃; the carrier gas is 99.999 percent high-purity helium, the flow rate is 1mL/min, and split-flow sample injection is not carried out; chromatographic column model HP-5 quartz capillary column of 30m × 0.32mm × 0.25 μm, temperature programmed conditions: the initial temperature of the column oven is 40 ℃, and then the temperature is increased to 80 ℃ at the speed of 1 ℃/min; then increasing the temperature to 250 ℃ at the speed of 20 ℃/min, and keeping the temperature for 10 min;
specifically, the MS detection conditions are: the ion source is an EI source, the electron energy is 70eV, the interface temperature is 250 ℃, the ion source temperature is 230 ℃, the mass scanning range is 30-540m/z, the solvent delay is 3min, and the full scanning mode is adopted.
The main fatty acid components of NC89 tobacco seed oil with antioxidant active ingredients are shown in table 1.
TABLE 1
1.2.2 determination of volatile component content:
detection conditions are as follows: nitrogen is taken as carrier gas, the flow rate is 2mL/min, the sample injection amount is 1 mu L, the split ratio is 1:29.5, and the temperature of a sample injection port and the temperature of a detector are 270 ℃ and 280 ℃ respectively; maintaining the initial column temperature at 100 deg.C for 3min, raising to 170 deg.C at 20 deg.C/min, maintaining for 10min, raising to 200 deg.C at 10 deg.C/min, maintaining for 5min, and raising to 230 deg.C at 2 deg.C/min, maintaining for 5 min; the rest is consistent with the determination method of the fatty acid content.
The volatile main components and the proportion of NC89 tobacco seed oil with antioxidant active ingredients are shown in Table 2.
TABLE 2
1.3 in vitro antioxidant Activity of NC89 tobacco seed oil with antioxidant active ingredient
Taking Vc as a positive control, and determining NC89 tobacco seed oil DPPH free radical scavenging capacity, ABTS free radical scavenging capacity and FRAP reducing capacity with different volume fractions (V/V) as antioxidant active ingredients.
1.3.1DPPH radical scavenging Capacity determination
The DPPH free radical scavenging capacity measuring method comprises the following steps: taking 0.20ml of tobacco seed oil and equal mass Vc, respectively adding 3.60, 4.80, 6.00, 7.20 and 8.40ml of 100 mu mol/L DPPH ethanol solution, reacting for 30min in a dark place, taking absolute ethyl alcohol as a blank control, measuring the light absorption value at 517nm, and calculating DPPH free radical clearance.
DPPH free radical clearance%A517-sample tubeA517) Comparison tubeA517×100％。
1.3.2ABTS radical scavenging Capacity determination
The ABTS free radical scavenging capacity determination method comprises the following steps: mixing 2.45mmol/L potassium persulfate solution and 7.00mmol/L ABTS stock solution equally, standing at room temperature in dark condition for 12-16h to obtain ABTS working solution; diluting ABTS working solution, and enabling OD of ABTS working solution to be at 37 DEG C734Obtaining ABTS determination solution when the ABTS determination solution is 0.70 +/-0.02; respectively mixing the ABTS determination solution with tobacco seed oil or Vc at volume ratio of 5%, 10%, 15%, 20%Mixing 25% and 30% to obtain mixed solution with different volume ratio, reacting at 37 deg.C in dark for 30min, measuring absorbance at 734nm, and recording the absorbance as ASample (I)(ii) a Replacing the sample with distilled water as blank control, and recording the absorbance of blank control at 734nm as ABlank space(ii) a Calculating the ABTS free radical clearance rate; ABTS free radical clearance is calculated by the formula of W% (1-A)Sample (I)/ABlank space)×100％。
1.3.3 measurement of Vc in vitro antioxidant Activity value by FRAP reduction Capacity measurement
Preparing an FRAP determination solution: 25mL of 0.3mol/L acetic acid buffer solution, 2.5mL of 10mmol/L TPTZ solution and 20mmol/LFeCl32.5mL of the solution is uniformly mixed and stored at 30 ℃ for later use;
and (3) standard curve preparation: taking FeSO with concentrations of 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0mmol/L respectively4Adding FRAP reagent 270 μ L into each 30 μ L of solution, incubating at 37 deg.C for 10min, measuring absorbance at 593nm with absorbance as abscissa and FeSO4Making a standard curve with the concentration as a vertical coordinate; FeSO required for FRAP value of sample to reach same absorbance4Expressed in millimoles, 1FRAP unit is 1.0mmol/L FeSO4The larger the FRAP value is, the higher the antioxidant activity is;
mixing FRAP determination solution with tobacco seed oil or Vc at volume ratio of 5%, 10%, 15%, 20%, 25%, 30% (total system 300 μ L) respectively, reacting at 30 deg.C in dark for 30min, using distilled water as blank control instead of sample, determining absorbance at 593nm, and calculating FRAP value according to standard curve.
The measurement results of the antioxidant activity index of NC89 tobacco seed oil are shown in Table 3.
TABLE 3
The test result shows that: NC89 tobacco seed oil has good antioxidant activity, and the antioxidant capacity is improved along with the increase of volume fraction. According to the table 3, the DPPH free radical scavenging force is greater than 50% when the volume fraction of NC89 tobacco seed oil is 3.23%, and the ABTS free radical scavenging force is greater than 50% when the volume fraction is 5%, which is obviously superior to the control; NC89 tobacco seed oil FRAP reduction capability is linearly related to volume fraction, and the FRAP reduction capability is strongest when the volume fraction is 30%, and is obviously better than a control.
The results show that NC89 tobacco seed oil has strong in-vitro antioxidant activity.
1.4 in vivo antioxidant Activity of NC89 tobacco seed oil with antioxidant active ingredient
H pair with NC89 tobacco seed oil2O2Inducing active oxygen content in HepG2 cell and NC89 tobacco seed oil pair H2O2Induction of HepG2 apoptosis value, NC89 tobacco seed oil vs H2O2And (3) inducing the GSH, SOD and CAT enzyme content in HepG2 cells to carry out comprehensive determination.
1.4.1 NC89 tobacco seed oil pairs H with antioxidant active ingredient2O2Inhibition assay for the Induction of Reactive Oxygen Species (ROS) production in HepG2 cells
The specific determination method comprises the following steps: the concentration of tobacco seed oil which is non-toxic to HepG2 cells was selected by MTT assay for subsequent testing. When HepG2 cells with certain concentration reach certain fusion degree after being cultured, the cells are pretreated by DMEM complete culture solution and DMEM complete culture medium containing NC89 tobacco seed oil with different concentrations, and then the DMEM complete culture solution and H are used2O2And (6) processing. H2O2After incubation, cells were collected, washed, added to DCFH-DA, incubated at 37 ℃ in the dark, and washed, and then the fluorescence intensity was measured using a flow cytometer.
The detection results are shown in fig. 1, where K: blank control group; y: a positive control group; m: a model group;#P<the concentration of the catalyst in the 0.05vs K group,*P<0.05vs group M.
From the test results of fig. 1, it can be seen that: h2O2Remarkably causes the generation of Reactive Oxygen Species (ROS) in HepG2 cells (625 +/-30 percent), and NC89 tobacco seed oilAnd positive control Vc of 10 mu g/mL can obviously inhibit H2O2The active oxygen ROS is generated in HepG2 cells, wherein the scavenging effect of NC89 tobacco seed oil on the active oxygen ROS is obviously better than that of Vc.
1.4.2 NC89 tobacco seed oil pairs H with antioxidant active ingredient2O2Inhibition assay for induction of apoptosis in HepG2 cells
The specific determination method comprises the following steps: when HepG2 cells with certain concentration reach certain fusion degree after being cultured, the cells are pretreated by DMEM complete culture solution and DMEM complete culture medium containing NC89 tobacco seed oil, and then the DMEM complete culture solution and H are used2O2And (6) processing. H2O2After incubation, the collected cells were washed, added with binding buffer, Annexin V-FITC and Propidium Iodide (PI), and incubated for 10min at room temperature in the absence of light. After the incubation was finished, the apoptosis was detected using a flow cytometer.
NC89 tobacco seed oil Pair H2O2Effect of inducing HepG2 apoptosis As shown in A of FIG. 2, NC89 tobacco seed oil on H2O2The effect of inducing HepG2 apoptosis rate is shown in fig. 2B. In fig. 2B, K: blank control group; m: a model group; y: and (4) a positive control group.#P<0.05vs K；*P<0.05vs group M.
As can be seen from the test results in fig. 2: h2O2Obviously causes the apoptosis of HepG2 cells, and the late apoptosis rate of the cells is 8.87%; after treatment with NC89 tobacco seed oil, the late apoptosis rate of the cells was 4.53% (fig. 2A). Therefore, NC89 seed oil was found to be effective in reducing the apoptosis rate and have a good inhibitory effect on apoptosis (fig. 2B).
1.4.3 NC89 tobacco seed oil pairs H with antioxidant active ingredient2O2Determination of the Effect of inducing the enzymatic Activity of GSH, SOD and CAT in HepG2 cells
The specific determination method comprises the following steps: when HepG2 cells with certain concentration reach certain fusion degree after being cultured, the cells are pretreated by DMEM complete culture solution and DMEM complete culture medium containing NC89 tobacco seed oil, and then the DMEM complete culture solution and H are used2O2And (6) processing. H2O2After incubation, the cells were lysed thoroughly and collected for intracellular assay of HepG2Glutathione peroxidase (GSH), superoxide dismutase (SOD) and Catalase (CAT).
NC89 tobacco seed oil pair H with antioxidant active ingredient2O2Induction of GSH, SOD and CAT enzyme activity effects in HepG2 cells is shown in figure 3, where K: blank control group; y: a positive control group; m: and (4) model groups.#P<The concentration of the catalyst in the 0.05vs K group,*P<0.05vs group M; wherein: FIG. 3A is a graph showing the change in GSH enzyme activity, FIG. 3B is a graph showing the change in SOD enzyme activity, and FIG. 3C is a graph showing the change in CAT enzyme activity.
As can be seen from the test results in fig. 3: h in comparison with blank control2O2Significantly induce reduction of intracellular GSH, SOD and CAT (P)<0.05); NC89 tobacco seed oil treatment significantly induced increases in intracellular GSH, SOD and CAT (P)<0.05、P<0.01). NC89 tobacco seed oil has good cell oxidation resistance, and the higher the concentration, the better the effect, can play a good cell protection role.
The results show that the NC89 tobacco seed oil has stronger in-vivo antioxidant activity.
1.5 application of NC89 tobacco seed oil with antioxidant active ingredient
By combining the fatty acid content and the volatile component content of the NC89 tobacco seed oil measured in the step 1.2, the in-vitro antioxidant activity value of the NC89 tobacco seed oil in the step 1.3 and the in-vivo antioxidant activity value of the NC89 tobacco seed oil in the step 1.4, the NC89 tobacco seed oil has stronger antioxidant activity. The NC89 tobacco seed oil can be added into edible oil or health products according to the requirements, and can effectively improve the antioxidant activity in the edible oil or health products.
Example 2
In this example, Baoshan No. 4 in aromatic tobacco was used as an example.
2.1A method for preparing Baoshan No. 4 variety tobacco seed oil with antioxidant active ingredients comprises the following steps:
(1) selecting raw materials: collecting mature tobacco capsule No. 4 Baoshan, and airing until the tobacco capsule No. 4 Baoshan is dried; threshing tobacco capsules, winnowing to obtain plump Baoshan No. 4 tobacco seeds, cleaning the plump tobacco seeds, and drying until the moisture content is reduced to 9% for later use;
(2) grinding the dried Baoshan No. 4 plump tobacco seeds, and leaching for 8 hours at 84 ℃ by using n-hexane as a solvent according to a material-liquid ratio of 1:18 to obtain Baoshan No. 4 tobacco seed primary oil;
(3) adding yeast powder biological adsorbent into the primary oil of Baoshan No. 4 tobacco seeds, and placing in a constant-temperature shaking table for oscillation adsorption reaction for 6 hours; the mass ratio of the yeast powder to the tobacco seed primary oil is 1: 20;
(4) after the reaction is finished, separating and filtering by using a 0.32 mu m microporous filter membrane to remove yeast powder to obtain a semi-finished product of Baoshan No. 4 tobacco seed oil;
(5) putting the semi-finished product of Baoshan No. 4 tobacco seed oil into a molecular distillation apparatus, wherein the feeding speed of the molecular distillation apparatus is 50mL/h, and the rotating speed of a rotor is 120 r/min; performing first molecular distillation at 81 deg.C under 100Pa, and condensing to obtain first fraction of tobacco seed oil L1 as first-stage light component; continuing performing secondary molecular distillation on the primary distillation residue at 101 ℃ under the condition of 10Pa, and condensing to obtain a second fraction which is the tobacco seed oil L2 of a secondary light component; continuously carrying out three-stage molecular distillation on the second distillation residue at the temperature of 121 ℃ and under the condition of 1Pa, and condensing to obtain tobacco seed oil L3 with a third fraction as a third-stage light component;
(6) and (4) combining the tobacco seed oil L1, the tobacco seed oil L2 and the tobacco seed oil L3 in the step (5) to obtain Baoshan No. 4 tobacco seed oil with an antioxidant active ingredient.
2.2, carrying out GC-MS detection on the Baoshan No. 4 tobacco seed oil with the antioxidant active ingredients, and determining the content of fatty acid and the content of volatile ingredients in the tobacco seed oil.
The method for measuring the fatty acid content and the method for measuring the volatile component content were the same as those in example 1.
The main fatty acid components of baoshan No. 4 tobacco seed oil with antioxidant active ingredients are shown in table 4.
Table 4 volatile main components and ratios of baoshan No. 4 tobacco seed oil having antioxidant active ingredients are shown in table 5.
TABLE 5
2.3 in vitro antioxidant Activity of Baoshan No. 4 tobacco seed oil with antioxidant active ingredients
Vc is used as a positive control, and DPPH free radical scavenging capacity, ABTS free radical scavenging capacity, hydroxyl free radical scavenging capacity and FRAP reducing capacity in Baoshan No. 4 tobacco seed oil with different volume fractions (V/V) and antioxidant active ingredients are determined.
The measurement methods of DPPH radical scavenging ability, ABTS radical scavenging ability and FRAP reducing ability were the same as those in example 1.
The method for measuring the hydroxyl radical scavenging capacity comprises the following steps:
reagent No. one: 0.02mol/L FeSO40.01mol/L salicylic acid 0.02mol/L H2O2＝1:2:1；
Reagent No. two: 0.02mol/L FeSO40.01mol/L salicylic acid and water are 1:2: 1.
Putting the tobacco seed oil or Vc into a 96-well plate according to the volume ratio of 5%, 10%, 15%, 20%, 25% and 30%, adding a No. 1 reagent into a sample group, adding a No. 2 reagent into a sample contrast group, replacing the sample with distilled water in a blank contrast group, adding the No. 1 reagent, supplementing a reaction system to 300 mu l by using the distilled water, reacting for 30min, measuring the absorbance at 510nm, and calculating the hydroxyl radical clearance. Hydroxyl radical clearance (%) ═ a3－(A1－A2)]/A3×100％(A1Absorbance of sample, A2Control Absorbance, A3Blank absorbance).
The measurement results of the antioxidant activity index of Baoshan No. 4 tobacco seed oil are shown in Table 6.
TABLE 6
The test result shows that: the Baoshan No. 4 tobacco seed oil has good integral antioxidant activity, and the antioxidant capacity is improved along with the increase of the volume fraction. According to table 6, when the volume fraction of baoshan No. 4 tobacco seed oil is 3.23%, the DPPH free radical scavenging force is greater than 50%, and when the volume fraction is 5%, the ABTS free radical scavenging force and hydroxyl free radical scavenging force are far greater than 50%, which is obviously superior to the control, and the effect is very good; the FRAP reducing ability of Baoshan No. 4 tobacco seed oil is linearly related to the volume fraction, and the FRAP reducing ability is strongest when the volume fraction is 30 percent and is obviously superior to that of a control.
The results show that the Baoshan No. 4 tobacco seed oil has strong in-vitro antioxidant activity.
2.4 in vivo antioxidant Activity of Baoshan No. 4 tobacco seed oil with antioxidant active ingredient
H pair by utilizing Baoshan No. 4 tobacco seed oil2O2Inducing active oxygen content in HepG2 cell and Paoshan No. 4 tobacco seed oil pair H2O2Inducing HepG2 cell apoptosis value and Baoshan No. 4 tobacco seed oil pair H2O2And (3) inducing the GSH, SOD and CAT enzyme content in HepG2 cells to carry out comprehensive determination.
2.4.1 Baoshan No. 4 tobacco seed oil pair H with antioxidant active ingredient2O2The inhibition of Reactive Oxygen Species (ROS) production in HepG2 cells was determined in accordance with example 1.
The results are shown in FIG. 4, where K: blank control group; m: a model group; y: and (4) a positive control group.#P<0.05vs K；*P<0.05vs group M;&P<0.05vs VCand (4) grouping.
As can be seen from the test results of fig. 4: h2O2Obviously causes the generation of Reactive Oxygen Species (ROS) in HepG2 cells (625 +/-30 percent), and the Baoshan No. 4 tobacco seed oil and the 10 mu g/mL positive control Vc can obviously inhibit H2O2The generated active oxygen ROS in HepG2 cells (160 +/-8 percent, 400 +/-10 percent), wherein the scavenging effect of Baoshan No. 4 tobacco seed oil on the active oxygen ROS is obviously better than that of Vc.
2.4.2 Baoshan No. 4 tobacco seed oil pair H with antioxidant active ingredient2O2Inhibition of apoptosis in HepG 2-inducing cells was determined in accordance with example 1.
The detection results are shown in FIG. 5, and the Paishan No. 4 tobacco seed oil pair H2O2The induction of HepG2 apoptosis is shown in A of FIG. 5, and Pao shan No. 4 tobacco seed oil is used for H2O2The rate of induction of HepG2 apoptosis is shown as B in fig. 5. In fig. 5B, K: blank control group; m: a model group; y: and (4) a positive control group.#P<0.05vs K；*P<0.05vs group M.
As can be seen from the test results in fig. 5: h2O2Obviously causes the apoptosis of HepG2 cells, and the late apoptosis rate of the cells is 8.87%; after treatment with baoshan No. 4 tobacco seed oil, the late apoptosis rate of the cells was 3.32% (fig. 5A). From this, it was found that the baoshan No. 4 tobacco seed oil had a good inhibitory effect on apoptosis (fig. 5B).
2.4.3 Baoshan No. 4 tobacco seed oil pair H with antioxidant active ingredient2O2Assay for the effect of inducing GSH, SOD and CAT enzyme activities in HepG2 cells, in a manner consistent with example 1.
Baoshan No. 4 tobacco seed oil pair H with antioxidant active ingredients2O2The effect of inducing the activity levels of GSH, SOD and CAT enzymes in HepG2 cells is shown in fig. 6, where K: blank control group; y: a positive control group; m: a model group;#P<the concentration of the catalyst in the 0.05vs K group,*P<0.05、**P<0.01vs group M. Wherein: FIG. 6A is a graph showing the change in GSH enzyme activity, FIG. 6B is a graph showing the change in SOD enzyme activity, and FIG. 6C is a graph showing the change in CAT enzyme activity.
As can be seen from the test results in fig. 6: h in comparison with blank control2O2Significantly induce reduction of intracellular GSH, SOD and CAT (P)<0.05); baoshan No. 4 tobacco seed oil treatment significantly induced increases in intracellular GSH, SOD and CAT (P)<0.05、P<0.01). The Baoshan No. 4 tobacco seed oil has good cell oxidation resistance, the higher the concentration is, the better the effect is, the oxidation resistance under 100ug/mL is equivalent to that of a positive control, and a good cell protection effect can be achieved.
The results show that the Baoshan No. 4 tobacco seed oil has stronger in-vivo antioxidant activity.
2.5 application of Baoshan No. 4 tobacco seed oil with antioxidant active ingredient
By combining the fatty acid content and the volatile component content of the Baoshan No. 4 tobacco seed oil measured in the step 2.2, the in-vitro antioxidant activity value of the Baoshan No. 4 tobacco seed oil in the step 2.3 and the in-vivo antioxidant activity value of the Baoshan No. 4 tobacco seed oil in the step 2.4, the Baoshan No. 4 tobacco seed oil has strong antioxidant activity. The Baoshan No. 4 tobacco seed oil can be added into edible oil or health products according to requirements, and can effectively improve the antioxidant activity of the edible oil or the health products.
Example 3
This example is an example of a Rustica variety in wild tobacco.
3.1A method for preparing tobacco seed oil of Rustica variety with antioxidant active ingredient comprises the following steps:
(1) selecting raw materials: collecting mature Rustica tobacco capsule, and airing until the Rustica tobacco capsule is dry; threshing tobacco capsules, winnowing to obtain plump Rustica tobacco seeds, cleaning the plump tobacco seeds, and drying until the moisture content is reduced to 9% for later use;
(2) grinding the dried full tobacco seeds of the Rustica, extracting for 9 hours at 86 ℃ by taking n-hexane as a solvent according to a material-liquid ratio of 1:18 to obtain primary oil of the Rustica tobacco seeds;
(3) adding yeast powder biological adsorbent into the rudica tobacco seed primary oil, and placing the rudica tobacco seed primary oil in a constant-temperature shaking table for oscillation adsorption reaction for 7 hours;
(4) after the reaction is finished, separating and filtering by using a 0.32 mu m microporous filter membrane to remove yeast powder to obtain a semi-finished product of the Rustica tobacco seed oil; the mass ratio of the yeast powder to the tobacco seed primary oil is 1: 20;
(5) putting the semi-finished product of the Rustica tobacco seed oil into a molecular distillation apparatus, wherein the feeding speed of the molecular distillation apparatus is 50mL/h, and the rotating speed of a rotor is 120 r/min; performing first molecular distillation at 79 ℃ under the condition of 100Pa, and obtaining smoke seed oil L1 with first fraction as a first-stage light component after condensation; continuing performing secondary molecular distillation on the first distillation residue at 99 ℃ under the condition of 10Pa, and condensing to obtain a second fraction which is the smoke seed oil L2 of a second-stage light component; continuously carrying out three-stage molecular distillation on the second distillation residue at 119 ℃ under the condition of 1Pa, and condensing to obtain tobacco seed oil L3 with a third fraction as a third-stage light component;
(6) and (4) combining the tobacco seed oil L1, the tobacco seed oil L2 and the tobacco seed oil L3 in the step (5) to obtain the Rustica tobacco seed oil with the antioxidant active ingredients.
3.2 carrying out GC-MS detection on the Rustica tobacco seed oil with the antioxidant active ingredients, and determining the content of fatty acid and the content of volatile ingredients in the tobacco seed oil.
The method for measuring the fatty acid content and the method for measuring the volatile component content were the same as those in example 1.
The main fatty acid components of Rustica tobacco seed oil with antioxidant active ingredients are shown in table 7.
TABLE 7
The volatile main components and the ratio of the Rustica tobacco seed oil with the antioxidant active ingredients are shown in table 9.
TABLE 8
3.3 in vitro antioxidant Activity of Rustica Smoke seed oil with antioxidant active ingredients
Vc is used as a positive control, and DPPH free radical scavenging capacity and FRAP reducing capacity of the Rustica tobacco seed oil with antioxidant active ingredients at different volume fractions (V/V) are determined.
The methods for measuring DPPH radical scavenging ability and FRAP reducing ability are the same as those in example 2.
The results of the determination of the antioxidant activity index of the Rustica tobacco seed oil are shown in Table 9.
TABLE 9
The test result shows that: the Rustica tobacco seed oil has good antioxidant activity, and the antioxidant capacity is improved along with the increase of volume fraction. According to the table 9, when the volume fraction of the Rustica tobacco seed oil is 2.33%, the DPPH free radical clearance is more than 50%, which is obviously better than that of the control; the FRAP reduction capability of the Rustica tobacco seed oil is linearly related to the volume fraction, and the FRAP reduction capability is strongest when the volume fraction is 30 percent and is obviously better than that of a control.
The results show that the Rustica tobacco seed oil has strong in-vitro antioxidant activity.
3.4 in vivo antioxidant Activity of Rustica tobacco seed oil with antioxidant active ingredients
H pair by using Rustica tobacco seed oil2O2Inducing active oxygen content in HepG2 cells and Rustica tobacco seed oil pair H2O2Induction of HepG2 apoptosis value, Rustica tobacco seed oil vs H2O2And comprehensively measuring the GSH and CAT enzyme contents in the induced HepG2 cells.
3.4.1 Rustica tobacco seed oil Pair H with antioxidant active ingredient2O2The inhibition of Reactive Oxygen Species (ROS) production in HepG2 cells was determined in accordance with example 1.
The detection results are shown in fig. 10, where K: blank control group; m: a model group; y: and (4) a positive control group.#P<0.05vs K；*P<0.05vs group M.
As can be seen from the test results of fig. 7: h2O2Obviously causes the generation of Reactive Oxygen Species (ROS) (625 +/-30%) in HepG2 cells, and both the Rustica tobacco seed oil and the positive control Vc of 10 mu g/mL can obviously inhibit H2O2The generated active oxygen ROS in HepG2 cells is caused, wherein the scavenging effect of the Rustica tobacco seed oil on the active oxygen ROS is better than that of Vc.
3.4.2 Rustica tobacco seed oil pair H with antioxidant active ingredient2O2Inhibition of apoptosis in HepG 2-inducing cells was determined in accordance with example 1.
The detection results are shown in FIG. 8, and the pairs of Rustica tobacco seed oil and H2O2Induction of HepG2 apoptosis was shown in FIG. 8A, and Rustica tobacco seed oil was used for H2O2The rate of induction of HepG2 apoptosis is shown in B in fig. 8. In fig. 8B, K: blank control group; m: and (4) model groups.
As can be seen from the test results in fig. 8: h2O2Obviously causes the apoptosis of HepG2 cells, and the late apoptosis rate of the cells is 8.87%; after the treatment of the Rustica tobacco seed oil, the late apoptosis rate of the cells was 4.81% (FIG. 8A). Therefore, the Rustica tobacco seed oil can effectively reduce the apoptosis rate and has a good inhibition effect on apoptosis (FIG. 8B).
3.4.3 Rustica tobacco seed oil Pair H with antioxidant active ingredient2O2Assay for the effect of inducing GSH and CAT enzyme activity in HepG2 cells, in a manner consistent with that of example 1.
Rustica tobacco seed oil pair H with antioxidant active ingredients2O2The effect of inducing the activity levels of GSH and CAT enzymes in HepG2 cells is shown in fig. 9, where K: blank control group; y: a positive control group; m: a model group; lower case English letters indicate significance of difference (P) between treatments<0.05). Wherein: FIG. 9A is a graph showing changes in GSH enzyme activity and FIG. 9B is a graph showing changes in CAT enzyme activity.
As can be seen from the test results in fig. 9: h in comparison with blank control2O2Remarkably induces the reduction of GSH and CAT in cells, and the treatment of the Rustica tobacco seed oil remarkably induces the increase of GSH and CAT in cells. The Rustica tobacco seed oil has good cell oxidation resistance, and the higher the concentration, the better the effect, and can play a good role in protecting cells.
The results show that the Rustica tobacco seed oil has strong in-vivo antioxidant activity.
3.5 application of Rustica tobacco seed oil with antioxidant active ingredient
And (3) combining the fatty acid content and the volatile component content of the Rustica tobacco seed oil measured in the step (3.2), the in-vitro antioxidant activity value of the Rustica tobacco seed oil in the step (3.3) and the in-vivo antioxidant activity value of the Rustica tobacco seed oil in the step (3.4), the Rustica tobacco seed oil is known to have strong antioxidant activity. The Rustica tobacco seed oil can be added into edible oil or health products according to the requirements, and can effectively improve the antioxidant activity of the edible oil or the health products.
Example 4
In this embodiment, the variety yunxue No. 2 in cigars is taken as an example.
4.1A method for preparing No. 2 Yunxue tobacco seed oil with antioxidant active ingredients comprises the following steps:
(1) selecting raw materials: collecting mature Yuxue No. 2 tobacco capsule, and airing until the Yuxue No. 2 tobacco capsule is dry; threshing tobacco capsules, winnowing to obtain plump tobacco seeds No. 2 Yunxue, cleaning the plump tobacco seeds, and drying until the moisture content is reduced to 9% for later use;
(2) grinding the dried Yuxue No. 2 plump tobacco seeds, and leaching for 9 hours at 86 ℃ by using n-hexane as a solvent according to a material-liquid ratio of 1:18 to obtain Yuxue No. 2 tobacco seed primary oil;
(3) adding yeast powder biological adsorbent into the Yuxue No. 2 tobacco seed primary oil, and placing the mixture in a constant-temperature shaking table for oscillation adsorption reaction for 7 hours; the mass ratio of the yeast powder to the tobacco seed primary oil is 1: 20;
(4) after the reaction is finished, separating and filtering by using a 0.32 mu m microporous filter membrane to remove yeast powder to obtain a semi-finished product of Baoyun snow No. 2 tobacco seed oil;
(5) putting the semi-finished product of the Yuxue No. 2 tobacco seed oil into a molecular distillation apparatus, wherein the feeding speed of the molecular distillation apparatus is 50mL/h, and the rotating speed of a rotor is 120 r/min; performing first molecular distillation at 79 deg.C under 100Pa, and condensing to obtain first fraction of oleum Nicotianae L1 as first-stage light component; continuing performing secondary molecular distillation on the first distillation residue at 99 ℃ under the condition of 10Pa, and condensing to obtain a second fraction which is the smoke seed oil L2 of a second-stage light component; continuously carrying out three-stage molecular distillation on the second distillation residue at 119 ℃ under the condition of 1Pa, and condensing to obtain tobacco seed oil L3 with a third fraction as a third-stage light component;
(6) and (4) combining the tobacco seed oil L1, the tobacco seed oil L2 and the tobacco seed oil L3 in the step (5) to obtain the Yunxue No. 2 tobacco seed oil with the antioxidant active ingredients.
3.2 carrying out GC-MS detection on the Yunxue No. 2 tobacco seed oil with the antioxidant active ingredients to determine the content of fatty acid and the content of volatile ingredients in the tobacco seed oil.
The method for measuring the fatty acid content and the method for measuring the volatile component content were the same as those in example 1.
The main fatty acid components of the yunxue No. 2 tobacco seed oil with antioxidant active ingredients are shown in table 10.
Table 10 the volatile main components and proportions of yunxue No. 2 tobacco seed oil having antioxidant active ingredients are shown in table 11.
TABLE 11
4.3 in vitro antioxidant Activity of Yuxue No. 2 Nicotiana seed oil with antioxidant active ingredient
Vc is used as a positive control, and DPPH free radical scavenging capacity, ABTS free radical scavenging capacity, hydroxyl free radical scavenging capacity and FRAP reducing capacity of the Yunxue No. 2 tobacco seed oil with different volume fractions (V/V) and antioxidant active ingredients are determined.
The measurement methods of DPPH radical scavenging ability, ABTS radical scavenging ability, hydroxyl radical scavenging ability and FRAP reducing ability were the same as those in example 2.
Through measurement, the measurement results of the antioxidant activity index of the Yuxue No. 2 tobacco seed oil are shown in table 12.
TABLE 12
The test result shows that: the Yuxue No. 2 tobacco seed oil has good integral antioxidant activity, and the antioxidant capacity is improved along with the increase of the volume fraction. According to table 12, when the volume fraction of the Yuxue No. 2 tobacco seed oil is 2.33%, the DPPH free radical scavenging force is greater than 50%, and when the volume fraction is 5%, the ABTS free radical scavenging force and hydroxyl free radical scavenging force are far greater than 50%, which is obviously superior to the control, and the effect is very good; FRAP reducing ability of the Yuxue No. 2 tobacco seed oil is linearly related to volume fraction, and the FRAP reducing ability is strongest when the volume fraction is 30%, and is obviously superior to that of a control.
The results show that the Yunxue No. 2 tobacco seed oil has strong in-vitro antioxidant activity.
4.4 in vivo antioxidant Activity of Yuxue No. 2 Nicotiana seed oil with antioxidant active ingredient
H pair by using Yuxue No. 2 tobacco seed oil2O2Inducing active oxygen content in HepG2 cells and the Paeonia yunnanensis No. 2 tobacco seed oil pair H2O2Inducing HepG2 cell apoptosis value and Yuxue No. 2 tobacco seed oil pair H2O2And comprehensively measuring the contents of GSH, SOD and CAT enzymes in the induced HepG2 cells.
4.4.1 Yunxue No. 2 tobacco seed oil pair H with antioxidant active ingredients2O2The inhibition of Reactive Oxygen Species (ROS) production in HepG2 cells was determined in accordance with example 1.
The detection results are shown in fig. 10, where K: blank control group; m: a model group; y: and (4) a positive control group.#P<0.05vs K；*P<0.05vs group M.
As can be seen from the test results of fig. 10: h2O2Obviously causes the generation of Reactive Oxygen Species (ROS) (625 +/-30%) in HepG2 cells, and the Yuxue No. 2 tobacco seed oil and the 10 mu g/mL positive control Vc can both obviously inhibit H2O2The generated active oxygen ROS in HepG2 cells is caused, wherein the eliminating effect of the Yunxue No. 2 tobacco seed oil on the active oxygen ROS is obviously better than that of Vc.
4.4.2 Yunxue No. 2 tobacco seed oil pair H with antioxidant active ingredients2O2Inhibition of apoptosis in HepG 2-inducing cells was determined in accordance with example 1.
The detection results are shown in FIG. 11, and the Paeonia yunnanensis No. 2 tobacco seed oil pair H2O2Induction of HepG2 apoptosis was performed as shown in A of FIG. 11, and the Nicotiana 2 seed oil was treated with H2O2The rate of induction of HepG2 apoptosis is shown in B in fig. 11. In fig. 11B, K: blank control group; m: and (4) model groups.
As can be seen from the test results in fig. 11: h2O2Obviously causes the apoptosis of HepG2 cells, and the late apoptosis rate of the cells is 8.87%; after the treatment of the Yuxue No. 2 tobacco seed oil, the late apoptosis rate of the cells is 3.48% (fig. 11A). From this, it was found that the yunxue No. 2 tobacco seed oil had a good inhibitory effect on apoptosis (fig. 11B).
4.4.3 Yunxue No. 2 tobacco seed oil pair H with antioxidant active ingredients2O2Assay for the effect of inducing GSH, SOD and CAT enzyme activities in HepG2 cells, in a manner consistent with example 1.
Yuxue No. 2 tobacco seed oil pair H with antioxidant active ingredients2O2Induction of GSH and CAT enzyme activity effects in HepG2 cells as shown in figure 12, K: blank control group; y: a positive control group; m: a model group;#P<the concentration of the catalyst in the 0.05vs K group,*P<0.05vs group M. Wherein: FIG. 12A is a graph showing the change in GSH enzyme activity, FIG. 12B is a graph showing the change in SOD enzyme activity, and FIG. 12C is a graph showing the change in CAT enzyme activity.
As can be seen from the test results in fig. 12: h in comparison with blank control2O2Significantly inducing the reduction of intracellular GSH, SOD and CAT; the treatment of the Yuxue No. 2 tobacco seed oil obviously induces the increase of GSH, SOD and CAT in cells. The Yuxue No. 2 tobacco seed oil has good cell oxidation resistance, and has good cell protection effect when the concentration is higher.
The results show that the Yunxue No. 2 tobacco seed oil has strong in-vivo antioxidant activity.
4.5 application of Yuxue No. 2 tobacco seed oil with antioxidant active ingredient
By combining the fatty acid content and the volatile component content of the Yuxue No. 2 tobacco seed oil measured in the step 4.2, the in vitro antioxidant activity value of the Yuxue No. 2 tobacco seed oil in the step 4.3 and the in vivo antioxidant activity value of the Yuxue No. 2 tobacco seed oil in the step 4.4, the Yuxue No. 2 tobacco seed oil is known to have strong antioxidant activity. The Yuxue No. 2 tobacco seed oil can be added into edible oil or health products according to the requirements, and can effectively improve the antioxidant activity of the edible oil or the health products.
Example 5
The embodiment takes the NC89 variety in the flue-cured tobacco as an example.
5.1A preparation method of NC89 tobacco seed oil with antioxidant active ingredients comprises the following steps:
(1) selecting raw materials: collecting mature NC89 tobacco capsule, and air-drying until NC89 tobacco capsule is dried; threshing tobacco capsules, winnowing to obtain plump NC89 tobacco seeds, cleaning the plump tobacco seeds, and drying until the moisture content is reduced to 8% for later use;
(2) grinding the dried full NC89 tobacco seeds, and leaching for 8 hours at 84 ℃ by using n-hexane as a solvent according to a material-liquid ratio of 1:18 to obtain NC89 tobacco seed primary oil;
(3) adding yeast powder biological adsorbent into NC89 tobacco seed primary oil, placing in a constant temperature shaking table, and performing oscillation adsorption reaction for 8 h; the mass ratio of the yeast powder to the tobacco seed primary oil is 1: 20;
(4) after the reaction is finished, separating and filtering by using a 0.32 mu m microporous filter membrane to remove yeast powder to obtain a semi-finished product of NC89 tobacco seed oil;
(5) placing the NC89 tobacco seed oil semi-finished product into a molecular distillation apparatus, wherein the feeding speed of the molecular distillation apparatus is 50mL/h, and the rotating speed of a rotor is 120 r/min; performing first molecular distillation at 79 deg.C under 100Pa, and condensing to obtain first fraction of oleum Nicotianae L1 as first-stage light component; continuing performing secondary molecular distillation on the first distillation residue at 99 ℃ under the condition of 10Pa, and condensing to obtain a second fraction which is the smoke seed oil L2 of a second-stage light component; continuously carrying out three-stage molecular distillation on the second distillation residue at the temperature of 119 ℃ and under the condition of 1Pa, and condensing to obtain tobacco seed oil L3 with a third fraction as a three-stage light component;
(6) and (3) combining the tobacco seed oil L1, the tobacco seed oil L2 and the tobacco seed oil L3 to obtain the NC89 tobacco seed oil with antioxidant active ingredients.
5.2, performing GC-MS detection on NC89 tobacco seed oil with antioxidant active ingredients to determine the content of fatty acid and the content of volatile ingredients in the tobacco seed oil.
5.2.1 determination method of fatty acid content: and (3) extracting and enriching NC89 tobacco seed oil samples by adopting a headspace solid phase microextraction method (HS-SPME), wherein the model of the extraction fiber head is 50/30 mu m DVB/CAR/PDMS. Before each use of the extraction fiber, the extraction fiber needs to be aged for 3min at a GC-MS injection port of 240 ℃. Accurately weighing 3mL NC89 tobacco seed oil with antioxidant active ingredient, adding into 20mL headspace bottle, immediately sealing, and extracting at 40 deg.C for 20 min. After extraction, the solid phase micro-extraction fiber needle is taken out, and then inserted into a GC injection port for desorption (3min, 240 ℃) and subsequent GC-MS analysis.
Specifically, the GC detection conditions were: the temperature of a sample inlet is 240 ℃; the carrier gas is 99.999 percent high-purity helium, the flow rate is 1mL/min, and split-flow sample injection is not carried out; chromatographic column model HP-5 quartz capillary column of 30m × 0.32mm × 0.25 μm, temperature programmed conditions: the initial temperature of the column oven is 40 ℃, and then the temperature is increased to 80 ℃ at the speed of 1 ℃/min; then increasing the temperature to 250 ℃ at the speed of 20 ℃/min, and keeping the temperature for 10 min;
specifically, the MS detection conditions are: the ion source is an EI source, the electron energy is 70eV, the interface temperature is 250 ℃, the ion source temperature is 230 ℃, the mass scanning range is 30-540m/z, the solvent delay is 3min, and the full scanning mode is adopted.
The main fatty acid components of NC89 tobacco seed oil with antioxidant active ingredients are shown in table 13.
Watch 13
5.2.2 determination of volatile component content:
detection conditions are as follows: nitrogen is taken as carrier gas, the flow rate is 2mL/min, the sample injection amount is 1 mu L, the split ratio is 1:29.5, and the temperature of a sample injection port and the temperature of a detector are 270 ℃ and 280 ℃ respectively; maintaining the initial column temperature at 100 deg.C for 3min, raising to 170 deg.C at 20 deg.C/min, maintaining for 10min, raising to 200 deg.C at 10 deg.C/min, maintaining for 5min, and raising to 230 deg.C at 2 deg.C/min, maintaining for 5 min; the rest is consistent with the determination method of the fatty acid content.
The volatile main components and the proportion of NC89 tobacco seed oil with antioxidant active ingredients are shown in table 14.
TABLE 14
5.3 in vitro antioxidant Activity of NC89 tobacco seed oil with antioxidant active ingredient
Taking Vc as a positive control, and determining NC89 tobacco seed oil DPPH free radical scavenging capacity, ABTS free radical scavenging capacity and FRAP reducing capacity with different volume fractions (V/V) as antioxidant active ingredients.
5.3.1DPPH radical scavenging Capacity determination
The DPPH free radical scavenging capacity measuring method comprises the following steps: taking 0.20ml of tobacco seed oil and equal mass Vc, respectively adding 3.60, 4.80, 6.00, 7.20 and 8.40ml of 100 mu mol/L DPPH ethanol solution, reacting for 30min in a dark place, taking absolute ethyl alcohol as a blank control, measuring the light absorption value at 517nm, and calculating DPPH free radical clearance.
DPPH free radical clearance%A517-sample tubeA517) Comparison tubeA517×100％。
5.3.2ABTS free radical scavenging assay
The ABTS free radical scavenging capacity determination method comprises the following steps: mixing 2.45mmol/L potassium persulfate solution and 7.00mmol/L ABTS stock solution equally, standing at room temperature in dark condition for 12-16h to obtain ABTS working solution; diluting ABTS working solution, and enabling OD of ABTS working solution to be at 37 DEG C734Obtaining ABTS determination solution when the ABTS determination solution is 0.70 +/-0.02; mixing ABTS determination solution with tobacco seed oil or Vc at volume ratio of 5%, 10%, 15%, 20%, 25%, and 30% respectively to obtain mixed solution with different volume ratio, reacting the mixed solution with different volume ratio at 37 deg.C in dark for 30min, and determining different volume ratiosThe absorbance of the mixed solution at 734nm is recorded as ASample(s)(ii) a Replacing the sample with distilled water as blank control, and recording the absorbance of blank control at 734nm as ABlank space(ii) a Calculating the ABTS free radical clearance rate; ABTS free radical clearance is calculated by the formula of W% (1-A)Sample(s)/ABlank space)×100％。
5.3.3 measurement of Vc in vitro antioxidant Activity value by FRAP reduction Capacity measurement
Preparing an FRAP determination solution: 25mL of 0.3mol/L acetic acid buffer solution, 2.5mL of 10mmol/L TPTZ solution and 20mmol/LFeCl32.5mL of the solution is uniformly mixed and stored at 30 ℃ for later use;
and (3) standard curve preparation: taking FeSO with concentrations of 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0mmol/L respectively4Adding FRAP reagent 270 μ L into each 30 μ L of solution, incubating at 37 deg.C for 10min, measuring absorbance at 593nm with absorbance as abscissa and FeSO4Making a standard curve with the concentration as a vertical coordinate; FeSO required for FRAP value of sample to reach same absorbance4Expressed in millimoles, 1FRAP unit is 1.0mmol/L FeSO4The larger the FRAP value is, the higher the antioxidant activity is;
mixing FRAP determination solution with tobacco seed oil or Vc at volume ratio of 5%, 10%, 15%, 20%, 25%, 30% (total system 300 μ L) respectively, reacting at 30 deg.C in dark for 30min, using distilled water as blank control instead of sample, determining absorbance at 593nm, and calculating FRAP value according to standard curve.
The results of measuring the antioxidant activity index of NC89 smoke seed oil are shown in table 15.
Watch 15
The test result shows that: NC89 tobacco seed oil has good antioxidant activity, and the antioxidant capacity is improved along with the increase of volume fraction. According to the table 15, the DPPH free radical scavenging force is greater than 50% when the NC89 tobacco seed oil volume fraction is 3.23%, and the ABTS free radical scavenging force is greater than 50% when the volume fraction is 5%, which is obviously superior to the control; NC89 tobacco seed oil FRAP reduction capability is linearly related to volume fraction, and the FRAP reduction capability is strongest when the volume fraction is 30%, and is obviously better than a control.
The results show that NC89 tobacco seed oil has strong in-vitro antioxidant activity.
5.4 in vivo antioxidant Activity of NC89 tobacco seed oil with antioxidant active ingredient
H pair with NC89 tobacco seed oil2O2Inducing active oxygen content in HepG2 cell and NC89 tobacco seed oil pair H2O2Induction of HepG2 apoptosis value, NC89 tobacco seed oil vs H2O2And (3) inducing the GSH, SOD and CAT enzyme content in HepG2 cells to carry out comprehensive determination.
5.4.1 NC89 tobacco seed oil pairs H with antioxidant active ingredient2O2Inhibition assay for the Induction of Reactive Oxygen Species (ROS) production in HepG2 cells
The specific determination method comprises the following steps: the concentration of tobacco seed oil which is non-toxic to HepG2 cells was selected by MTT assay for subsequent testing. When HepG2 cells with certain concentration reach certain fusion degree after being cultured, the cells are pretreated by DMEM complete culture solution and DMEM complete culture medium containing NC89 tobacco seed oil with different concentrations, and then the DMEM complete culture solution and H are used2O2And (6) processing. H2O2After incubation, cells were collected, washed, added to DCFH-DA, incubated at 37 ℃ in the dark, and washed, and then the fluorescence intensity was measured using a flow cytometer.
5.4.2 NC89 tobacco seed oil pairs H with antioxidant active ingredient2O2Inhibition assay for inducing apoptosis of HepG2
The specific determination method comprises the following steps: when HepG2 cells with certain concentration reach certain fusion degree after being cultured, the cells are pretreated by DMEM complete culture solution and DMEM complete culture medium containing NC89 tobacco seed oil, and then the DMEM complete culture solution and H are used2O2And (6) processing. H2O2After incubation, the collected cells were washed, added with binding buffer, Annexin V-FITC and Propidium Iodide (PI), and incubated for 10min at room temperature in the absence of light. After the incubation was finished, the apoptosis was detected using a flow cytometer.
5.4.3N with antioxidant active ingredientC89 tobacco seed oil Pair H2O2Determination of the Effect of inducing the enzymatic Activity of GSH, SOD and CAT in HepG2 cells
The specific determination method comprises the following steps: when HepG2 cells with certain concentration reach certain fusion degree after being cultured, the cells are pretreated by DMEM complete culture solution and DMEM complete culture medium containing NC89 tobacco seed oil, and then the DMEM complete culture solution and H are used2O2And (6) processing. H2O2After incubation, the cells were lysed thoroughly and collected for determination of glutathione peroxidase (GSH), superoxide dismutase (SOD) and Catalase (CAT) levels in HepG2 cells.
The result shows that NC89 tobacco seed oil has stronger in-vivo antioxidant activity capability.
5.5 application of NC89 tobacco seed oil with antioxidant active ingredient
By combining the fatty acid content and the volatile component content of the NC89 tobacco seed oil measured in the step 5.2, the in-vitro antioxidant activity value of the NC89 tobacco seed oil in the step 5.3 and the in-vivo antioxidant activity value of the NC89 tobacco seed oil in the step 5.4, the NC89 tobacco seed oil has stronger antioxidant activity. NC89 tobacco seed oil can be added into edible oil or health products as required, and can effectively improve the antioxidant activity of the edible oil or health products.
Example 6
The embodiment takes the NC89 variety in the flue-cured tobacco as an example.
6.1A preparation method of NC89 tobacco seed oil with antioxidant active ingredients comprises the following steps:
(1) selecting raw materials: collecting mature NC89 tobacco capsule, and air-drying until NC89 tobacco capsule is dried; threshing tobacco capsules, winnowing to obtain plump NC89 tobacco seeds, cleaning the plump tobacco seeds, and drying until the moisture content is reduced to 10% for later use;
(2) grinding the dried full NC89 tobacco seeds, and leaching for 9 hours at 86 ℃ by using n-hexane as a solvent according to a material-liquid ratio of 1:18 to obtain NC89 tobacco seed primary oil;
(3) adding yeast powder biological adsorbent into NC89 tobacco seed primary oil, placing in a constant temperature shaking table, and performing oscillation adsorption reaction for 8 h; the mass ratio of the yeast powder to the tobacco seed primary oil is 1: 20;
(4) after the reaction is finished, separating and filtering by using a 0.32 mu m microporous filter membrane to remove yeast powder to obtain a semi-finished product of NC89 tobacco seed oil;
(5) placing the NC89 tobacco seed oil semi-finished product into a molecular distillation apparatus, wherein the feeding speed of the molecular distillation apparatus is 50mL/h, and the rotating speed of a rotor is 120 r/min; performing first molecular distillation at 81 deg.C under 100Pa to obtain first fraction of oleum Nicotianae L1 as first-stage light component; continuing performing secondary molecular distillation on the primary distillation residues at the temperature of 101 ℃ and under the condition of 10Pa, and condensing to obtain a second fraction which is the smoke seed oil L2 of the secondary light component; continuously carrying out three-stage molecular distillation on the second distillation residue at the temperature of 121 ℃ and under the condition of 1Pa, and condensing to obtain tobacco seed oil L3 with a third fraction as a third-stage light component;
(6) and (3) combining the tobacco seed oil L1, the tobacco seed oil L2 and the tobacco seed oil L3 to obtain the NC89 tobacco seed oil with antioxidant active ingredients.
6.2 carrying out GC-MS detection on NC89 tobacco seed oil with antioxidant active ingredients to determine the content of fatty acid and the content of volatile ingredients in the tobacco seed oil.
6.2.1 determination method of fatty acid content: and (3) extracting and enriching NC89 tobacco seed oil samples by adopting a headspace solid phase microextraction method (HS-SPME), wherein the model of the extraction fiber head is 50/30 mu m DVB/CAR/PDMS. Before each use of the extraction fiber, the extraction fiber needs to be aged for 3min at a GC-MS injection port of 240 ℃. Accurately weighing 3mL NC89 tobacco seed oil with antioxidant active ingredient, adding into 20mL headspace bottle, immediately sealing, and extracting at 40 deg.C for 20 min. After extraction, the solid phase micro-extraction fiber needle is taken out, and then inserted into a GC injection port for desorption (3min, 240 ℃) and subsequent GC-MS analysis.
Specifically, the GC detection conditions were: the temperature of a sample inlet is 240 ℃; the carrier gas is 99.999 percent high-purity helium, the flow rate is 1mL/min, and split-flow sample injection is not carried out; chromatographic column model HP-5 quartz capillary column of 30m × 0.32mm × 0.25 μm, temperature programmed conditions: the initial temperature of the column oven is 40 ℃, and then the temperature is increased to 80 ℃ at the speed of 1 ℃/min; then increasing the temperature to 250 ℃ at the speed of 20 ℃/min, and keeping the temperature for 10 min;
specifically, the MS detection conditions are: the ion source is an EI source, the electron energy is 70eV, the interface temperature is 250 ℃, the ion source temperature is 230 ℃, the mass scanning range is 30-540m/z, the solvent delay is 3min, and the full scanning mode is adopted.
The main fatty acid components of NC89 tobacco seed oil with antioxidant active ingredients are shown in table 16.
TABLE 16
6.2.2 determination of volatile component content:
detection conditions are as follows: nitrogen is taken as carrier gas, the flow rate is 2mL/min, the sample injection amount is 1 mu L, the split ratio is 1:29.5, and the temperature of a sample injection port and the temperature of a detector are 270 ℃ and 280 ℃ respectively; maintaining the initial column temperature at 100 deg.C for 3min, raising to 170 deg.C at 20 deg.C/min, maintaining for 10min, raising to 200 deg.C at 10 deg.C/min, maintaining for 5min, and raising to 230 deg.C at 2 deg.C/min, maintaining for 5 min; the rest is consistent with the determination method of the fatty acid content.
The volatile main components and the proportion of NC89 tobacco seed oil having antioxidant active ingredients are shown in table 17.
TABLE 17
6.3 in vitro antioxidant Activity of NC89 tobacco seed oil with antioxidant active ingredient
Taking Vc as a positive control, and determining NC89 tobacco seed oil DPPH free radical scavenging capacity, ABTS free radical scavenging capacity and FRAP reducing capacity with different volume fractions (V/V) as antioxidant active ingredients.
6.3.1DPPH radical scavenging Capacity determination
The DPPH free radical scavenging capacity measuring method comprises the following steps: taking 0.20ml of tobacco seed oil and equal mass Vc, respectively adding 3.60, 4.80, 6.00, 7.20 and 8.40ml of 100 mu mol/L DPPH ethanol solution, reacting for 30min in a dark place, taking absolute ethyl alcohol as a blank control, measuring the light absorption value at 517nm, and calculating DPPH free radical clearance.
DPPH free radical scavenging ratio%(control tube)A517-sample tubeA517) Comparison tubeA517×100％。
6.3.2ABTS free radical scavenging assay
The ABTS free radical scavenging capacity determination method comprises the following steps: mixing 2.45mmol/L potassium persulfate solution and 7.00mmol/L ABTS stock solution equally, standing at room temperature in dark condition for 12-16h to obtain ABTS working solution; diluting ABTS working solution, and enabling OD of ABTS working solution to be at 37 DEG C734Obtaining ABTS determination solution when the ABTS determination solution is 0.70 +/-0.02; respectively and fully mixing the ABTS determination solution and tobacco seed oil or Vc according to the volume ratio of 5%, 10%, 15%, 20%, 25% and 30% to obtain mixed solutions with different volume ratios, carrying out dark reaction on the mixed solutions with different volume ratios at 37 ℃ for 30min, and then determining the light absorption values of the mixed solutions with different volume ratios at 734nm, wherein the light absorption value is marked as ASample (I)(ii) a Replacing the sample with distilled water as blank control, and recording the absorbance of blank control at 734nm as ABlank space(ii) a Calculating the ABTS free radical clearance rate; ABTS free radical clearance is calculated by the formula of W% (1-A)Sample (I)/ABlank space)×100％。
6.3.3 measurement of Vc in vitro antioxidant Activity value by FRAP reduction Capacity measurement
Preparing an FRAP determination solution: 25mL of 0.3mol/L acetate buffer, 2.5mL of 10mmol/L TPTZ solution, and 20mmol/LFeCl32.5mL of the solution is uniformly mixed and stored at 30 ℃ for later use;
and (3) standard curve preparation: taking FeSO with concentrations of 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0mmol/L respectively4Adding FRAP reagent 270 μ L into each 30 μ L of solution, incubating at 37 deg.C for 10min, measuring absorbance at 593nm with absorbance as abscissa and FeSO4Making a standard curve with the concentration as a vertical coordinate; FeSO required for FRAP value of sample to reach same absorbance4Expressed in millimoles, 1FRAP unit is 1.0mmol/L FeSO4The larger the FRAP value is, the higher the antioxidant activity is;
mixing FRAP determination solution with tobacco seed oil or Vc at volume ratio of 5%, 10%, 15%, 20%, 25%, 30% (total system 300 μ L) respectively, reacting at 30 deg.C in dark for 30min, using distilled water as blank control instead of sample, determining absorbance at 593nm, and calculating FRAP value according to standard curve.
The results of measuring the antioxidant activity index of NC89 smoke seed oil are shown in table 18.
The test result shows that: NC89 tobacco seed oil has good antioxidant activity, and the antioxidant capacity is improved along with the increase of volume fraction. According to table 18, the DPPH radical scavenging force is greater than 50% at a NC89 tobacco seed oil volume fraction of 3.23%, and the ABTS radical scavenging force is much greater than 50% at a NC89 tobacco seed oil volume fraction of 5%, which is significantly better than the control; NC89 tobacco seed oil FRAP reduction capability is linearly related to volume fraction, and the FRAP reduction capability is strongest when the volume fraction is 30%, and is obviously better than a control.
The results show that NC89 tobacco seed oil has strong in-vitro antioxidant activity.
6.4 in vivo antioxidant Activity of NC89 tobacco seed oil with antioxidant active ingredient
H pair with NC89 tobacco seed oil2O2Inducing active oxygen content in HepG2 cell and NC89 tobacco seed oil pair H2O2Induction of HepG2 apoptosis value, NC89 tobacco seed oil vs H2O2And (3) inducing the GSH, SOD and CAT enzyme content in HepG2 cells to carry out comprehensive determination.
6.4.1 NC89 tobacco seed oil pairs H with antioxidant active ingredient2O2Measurement of inhibition of Reactive Oxygen Species (ROS) production in HepG2 cells
The specific determination method comprises the following steps: the concentration of tobacco seed oil which is non-toxic to HepG2 cells was selected by MTT assay for subsequent testing. When HepG2 cells with certain concentration reach certain fusion degree after being cultured, the cells are pretreated by DMEM complete culture solution and DMEM complete culture medium containing NC89 tobacco seed oil with different concentrations, and then the DMEM complete culture solution and H are used2O2And (6) processing. H2O2After incubation, collecting cells, washing, adding DCFH-DA, incubating at 37 ℃ in a dark place, washing, and measuring fluorescence by using a flow cytometerLight intensity.
6.4.2 NC89 tobacco seed oil pairs H with antioxidant active ingredient2O2Inhibition assay for inducing apoptosis of HepG2
The specific determination method comprises the following steps: when HepG2 cells with certain concentration reach certain fusion degree after being cultured, the cells are pretreated by DMEM complete culture solution and DMEM complete culture medium containing NC89 tobacco seed oil, and then the DMEM complete culture solution and H are used2O2And (6) processing. H2O2After incubation, the collected cells were washed, added with binding buffer, Annexin V-FITC, and Propidium Iodide (PI), and incubated for 10min at room temperature in the dark. After the incubation was finished, the apoptosis was detected using a flow cytometer.
6.4.3 NC89 tobacco seed oil pairs H with antioxidant active ingredient2O2Determination of the Effect of inducing the enzymatic Activity of GSH, SOD and CAT in HepG2 cells
The specific determination method comprises the following steps: when HepG2 cells with certain concentration reach certain fusion degree after being cultured, the cells are pretreated by DMEM complete culture solution and DMEM complete culture medium containing NC89 tobacco seed oil, and then the DMEM complete culture solution and H are used2O2And (6) processing. H2O2After incubation, the cells were lysed well and the cells were harvested for determination of glutathione peroxidase (GSH), superoxide dismutase (SOD) and Catalase (CAT) levels in HepG2 cells.
The result shows that the NC89 tobacco seed oil has stronger in-vivo antioxidant activity.
6.5 application of NC89 tobacco seed oil with antioxidant active ingredient
By combining the fatty acid content and the volatile component content of the NC89 tobacco seed oil measured in the step 6.2, the in-vitro antioxidant activity value of the NC89 tobacco seed oil in the step 6.3 and the in-vivo antioxidant activity value of the NC89 tobacco seed oil in the step 6.4, the NC89 tobacco seed oil has stronger antioxidant activity. NC89 tobacco seed oil can be added into edible oil or health products as required, and can effectively improve the antioxidant activity of the edible oil or health products.
The cytotoxicity of the tobacco seed oil obtained in examples 1 to 6 was measured by the following specific method:
(1) cell digestion: RAW 264.7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 1% diabody (penicillin (100U/mL), streptomycin (100mg/mL) at 37 ℃ under 5% CO295% air condition cell incubator.
(2) Subjecting RAW 264.7 cells in logarithmic growth phase to digestion and centrifugation according to the digestion method in (1) to obtain RAW 264.7 cells, counting by a cell counter, and adjusting cell density to 1.0 × 105And (4) one cell per ml, performing adherent growth in 96 holes, and treating the cells with 400, 200, 100, 50 and 25 mu g/ml of tobacco seed oil for 20 hours after adherent treatment for 24 hours. After the completion of the culture, the medium was removed, and 200. mu.L of MTT reagent (0.50 mg/mL) was added thereto to continue the culture. After the cells are treated for 4 hours, sucking away the supernatant, adding 200 mu L DMSO again, shaking fully in the dark to dissolve the crystal violet completely, measuring the 490nm light absorption value by using an enzyme-labeling instrument, and calculating the cell survival rate.
Cell survival (%) ═ (OD)Sample set-ODBlank group)/(ODControl group-ODBlank group)×100％。
The tobacco seed oil prepared in examples 1-6 has no significant cytotoxicity to RAW 264.7 cells; therefore, it can be taken.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A preparation method of tobacco seed oil with antioxidant active ingredients is characterized by comprising the following steps: the method comprises the following steps:
(1) selecting raw materials: collecting mature tobacco capsule, and air-drying until the tobacco capsule is dry; threshing tobacco capsules, winnowing to obtain plump tobacco seeds, cleaning the plump tobacco seeds, and drying until the moisture content is reduced to 8% -10% for later use;
(2) extracting the plump tobacco seeds dried in the step (1) by using a Soxhlet extraction method to obtain tobacco seed primary oil;
(3) adding a biological adsorbent into the tobacco seed primary oil obtained in the step (2), and placing the mixture in a constant-temperature shaking table for oscillation adsorption reaction for 6-8 hours;
(4) after the reaction in the step (3) is finished, separating and filtering the mixture by using a microporous filter membrane to remove the biological adsorbent to obtain a semi-finished product of the tobacco seed oil;
(5) putting the semi-finished product of the tobacco seed oil in the step (4) into a molecular distillation instrument, carrying out first molecular distillation at 79-81 ℃ under the condition of 100Pa, and obtaining tobacco seed oil L1 with first fraction as a first-stage light component after condensation; continuing to perform secondary molecular distillation on the first distillation residue at 99-101 ℃ under the condition of 10Pa, and condensing to obtain second fraction which is smoke seed oil L2 of a second-stage light component; continuously carrying out three-stage molecular distillation on the second distillation residue at the temperature of 119-121 ℃ and under the condition of 1Pa, and condensing to obtain tobacco seed oil L3 with a third fraction as a three-stage light component;
(6) and (4) combining the tobacco seed oil L1, the tobacco seed oil L2 and the tobacco seed oil L3 in the step (5) to obtain the tobacco seed oil with the antioxidant active ingredients.
2. The method for preparing the tobacco seed oil with the antioxidant active ingredient according to claim 1, wherein the method comprises the following steps: the Soxhlet extraction method in the step (2) is preferably to grind the dried plump tobacco seeds, take n-hexane as a solvent, and extract for 8-9h at 84-86 ℃ according to the material-liquid ratio of 1: 18.
3. The method for preparing the tobacco seed oil with the antioxidant active ingredient according to claim 1, wherein the method comprises the following steps: the biological adsorbent in the step (3) is preferably yeast powder, and the mass ratio of the yeast powder to the primary tobacco seed oil is 1: 20.
4. The method for preparing the tobacco seed oil with the antioxidant active ingredient according to claim 1, wherein the method comprises the following steps: the pore diameter of the microporous filter membrane in the step (4) is 0.32 mu m.
5. The method for preparing the tobacco seed oil with the antioxidant active ingredient according to claim 1, wherein the method comprises the following steps: the feeding speed of the molecular distillation instrument in the step (5) is 50mL/h, and the rotating speed of a rotor is 120 r/min.
6. A method for evaluating the antioxidant activity of tobacco seed oil is characterized by comprising the following steps: the method comprises the following steps:
s1: performing GC-MS detection on the tobacco seed oil with the antioxidant active ingredients according to any one of claims 1 to 5 to detect the content of fatty acid and the content of volatile ingredients in the tobacco seed oil with the antioxidant active ingredients;
s2: measuring the in vitro antioxidant activity and the in vivo cell level antioxidant activity of Vc by taking Vc as a positive control to obtain an in vitro antioxidant activity value and an in vivo cell level antioxidant activity value of Vc;
s3: measuring the in vitro antioxidant activity and the in vivo cell level antioxidant activity of the tobacco seed oil to obtain an in vitro antioxidant activity value and an in vivo cell level antioxidant activity value of the tobacco seed oil;
s4: comparing the Vc in-vitro antioxidant activity value in the step S2 with the in-vitro antioxidant activity value of the tobacco seed oil in the step S3 to obtain the in-vitro antioxidant activity capacity of the tobacco seed oil;
s5: comparing the Vc in-vivo cell level antioxidant activity value in the step S2 with the in-vivo cell level antioxidant activity value of the tobacco seed oil in the step S3 to obtain the in-vivo antioxidant activity capacity of the tobacco seed oil;
s6: and (4) evaluating the antioxidant activity of the tobacco seed oil by combining the in-vitro antioxidant activity of the tobacco seed oil in the step S4, the in-vivo antioxidant activity of the tobacco seed oil in the step S5, the fatty acid content and the volatile component content of the tobacco seed oil in the step S1.
7. The method for evaluating antioxidant activity of tobacco seed oil according to claim 6, wherein: the GC-MS detection conditions for the fatty acid content in step S1 were:
GC detection conditions are as follows: the temperature of a sample inlet is 240 ℃; the carrier gas is 99.999 percent high-purity helium, the flow rate is 1mL/min, and split-flow sample injection is not carried out; chromatographic column model HP-5 quartz capillary column of 30m × 0.32mm × 0.25 μm, temperature programmed conditions: the initial temperature of the column oven is 40 ℃, and then the temperature is increased to 80 ℃ at the speed of 1 ℃/min; then increasing the temperature to 250 ℃ at the speed of 20 ℃/min, and keeping the temperature for 10 min;
MS detection conditions: the ion source is an EI source, the electron energy is 70eV, the interface temperature is 250 ℃, the ion source temperature is 230 ℃, the mass scanning range is 30-540m/z, the solvent delay is 3min, and the full scanning mode is adopted.
8. The method for evaluating antioxidant activity of tobacco seed oil according to claim 6, wherein: the determination methods of the in-vitro antioxidant activity value of Vc in the step S2 and the in-vitro antioxidant activity value of the tobacco seed oil in the step S3 are as follows: the comprehensive measurement is carried out by using two or more of DPPH free radical scavenging ability, ABTS free radical scavenging ability, hydroxyl free radical scavenging ability and FRAP reducing ability.
9. The method for evaluating antioxidant activity of tobacco seed oil according to claim 6, wherein: the determination method of the in vivo antioxidant activity value of Vc in the step S2 and the in vivo antioxidant activity value of the tobacco seed oil in the step S3 is that: h pair by using tobacco seed oil2O2Inducing active oxygen content in HepG2 cells and tobacco seed oil pair H2O2Induction of HepG2 apoptosis value, tobacco seed oil pair H2O2And comprehensively measuring the content of GSH, SOD and CAT enzymes or the content of GSH and CAT enzymes in the induced HepG2 cells.
10. The application of the tobacco seed oil with the antioxidant active ingredient is characterized in that: the tobacco seed oil with the antioxidant active ingredients is added into edible oil or health care products after the antioxidant activity evaluation is qualified.
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