CN114525167A - Preparation method of tobacco seed oil with anti-inflammatory active ingredient, anti-inflammatory activity evaluation method and application - Google Patents

Preparation method of tobacco seed oil with anti-inflammatory active ingredient, anti-inflammatory activity evaluation method and application Download PDF

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CN114525167A
CN114525167A CN202210173537.2A CN202210173537A CN114525167A CN 114525167 A CN114525167 A CN 114525167A CN 202210173537 A CN202210173537 A CN 202210173537A CN 114525167 A CN114525167 A CN 114525167A
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tobacco seed
inflammatory
seed oil
tobacco
oil
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CN114525167B (en
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古吉
程桂广
郑昀晔
宋碧清
牛永志
郑方圆
张晓玉
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YUXI ZHONGYAN TOBACCO SEED CO Ltd
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YUXI ZHONGYAN TOBACCO SEED CO Ltd
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    • C—CHEMISTRY; METALLURGY
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Abstract

The invention discloses a preparation method of tobacco seed oil with an anti-inflammatory active ingredient, an anti-inflammatory activity evaluation method and application, wherein the preparation method of the tobacco seed oil with the anti-inflammatory active ingredient comprises the steps of selecting raw materials, and performing cold pressing on the raw materials to obtain tobacco seed primary oil; centrifuging the tobacco seed primary oil 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 h; 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 tertiary molecular distillation, and combining three fractions of the tobacco seed oil subjected to tertiary molecular distillation to obtain the high-purity tobacco seed oil with the anti-inflammatory active ingredients. The tobacco seed oil is added into vaseline to be used as anti-inflammatory cream after the anti-inflammatory activity is evaluated to be qualified, or added into beeswax to be used as lipstick for cheilitis, or directly used as anti-inflammatory essential oil to exert the anti-inflammatory activity.

Description

Preparation method of tobacco seed oil with anti-inflammatory active ingredient, anti-inflammatory activity evaluation method and application
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 anti-inflammatory active ingredient, an anti-inflammatory activity evaluation method and application.
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 that 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 physically squeezed tobacco seed oil cannot be finely applied. 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 anti-inflammatory activity, and a method for evaluating anti-inflammatory activity and application 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 anti-inflammatory active ingredient, the second purpose of the invention is to provide an evaluation method of the anti-inflammatory activity of the tobacco seed oil, and the third purpose of the invention is to provide the application of the tobacco seed oil with the anti-inflammatory active ingredient.
The first purpose of the invention is realized by a method for preparing the tobacco seed oil with the anti-inflammatory 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) crushing the plump tobacco seeds dried in the step (1), and then performing cold pressing to obtain cold pressed tobacco seed primary oil;
(3) centrifuging the tobacco seed primary oil in the step (2) at 7000-8000r/min, and then discarding solid phase substances to obtain the tobacco seed primary oil;
(4) adding a biological adsorbent into the tobacco seed primary oil in the step (3), and placing the mixture in a constant-temperature shaking table for oscillation adsorption reaction for 6-8 hours;
(5) after the reaction in the step (4) 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;
(6) putting the semi-finished product of the tobacco seed oil in the step (5) into a molecular distillation instrument, carrying out first molecular distillation at 90-100 ℃ under the condition of 100Pa, and obtaining tobacco 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 the temperature of 100-110 ℃ and under the condition of 10Pa, and condensing to obtain a second fraction which is the smoke seed oil L2 of the second-stage light component; continuously carrying out three-stage molecular distillation on the second distillation residue at the temperature of 120-130 ℃ and under the condition of 1Pa, and condensing to obtain smoke seed oil L3 with a third fraction as a three-stage light component;
(7) and (4) combining the tobacco seed oil L1, the tobacco seed oil L2 and the tobacco seed oil L3 in the step (6) to obtain the tobacco seed oil with the anti-inflammatory active ingredients.
Preferably, the cold pressing in the step (2) is that the cold pressing is carried out at the temperature of below 70 ℃ after the full tobacco seeds are crushed to the granularity of 60-70 meshes.
Preferably, the biosorbent in step (4) is yeast powder, and the mass ratio of the yeast powder to the tobacco seed crude oil is 1: 20.
Preferably, the pore size of the microporous filter membrane in the step (5) is 0.32 μm.
Preferably, the feeding speed of the molecular distillation instrument in the step (6) is 50mL/h, and the rotating speed of a rotor is 120 r/min.
The second object of the invention is realized by a method for evaluating the anti-inflammatory activity of the tobacco seed oil, which comprises the following steps:
s1: subjecting the tobacco seed oil with anti-inflammatory active ingredients of any one of claims 1-5 to GC-MS detection to detect the content of fatty acids and volatile components in the tobacco seed oil with anti-inflammatory active ingredients;
s2: measuring the content of NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in LPS-induced RAW264.7 cells by using 10 mu M dexamethasone as a positive control;
s3: selecting 25-100 mu g/ml of tobacco seed oil, and determining the content of NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in the RAW264.7 cells induced by LPS by the tobacco seed oil with different concentrations;
s4: comparing the content of NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in LPS-induced RAW264.7 cells by 10 mu M dexamethasone in the step S2 with the content of NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in LPS-induced RAW264.7 cells by tobacco seed oil with different corresponding concentrations in the step S3, and determining the anti-inflammatory activity capability of the tobacco seed oil;
s5: and comprehensively evaluating the anti-inflammatory activity of the tobacco seed oil by combining the anti-inflammatory activity of the tobacco seed oil determined in the step S4, the fatty acid content and the volatile component content of the tobacco seed oil determined 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; increasing to 250 deg.C at a rate of 20 deg.C/min, and maintaining 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, in step S2 or step S3, the content of NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in the LPS-induced RAW264.7 cells is determined by dexamethasone or tobacco seed oil:
culturing RAW264.7 cells and inoculating the cells in a culture medium, and removing the culture medium after the RAW264.7 cells grow in a complete adherent manner; adding 25-100 μ g/ml tobacco seed oil and 10 μ M dexamethasone to treat RAW264.7 cells, removing supernatant after treatment, washing RAW264.7 cells with PBS, adding LPS again to continue culturing; after the culture is finished, collecting lysate, and determining the content of NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in RAW264.7 cells according to the kit specification.
The third purpose of the invention is realized by the application of the tobacco seed oil with the anti-inflammatory active ingredient, wherein the tobacco seed oil with the anti-inflammatory active ingredient is added into vaseline as an anti-inflammatory cream after the anti-inflammatory activity is qualified, or is added into beeswax as a cheilitis lipstick, or is directly used as an anti-inflammatory essential oil.
Compared with the prior art, the invention has the following technical effects:
1. the invention extracts the tobacco seed primary oil by cold pressing tobacco seeds, and then carries out three times of molecular distillation on the tobacco seed semi-finished oil which is adsorbed by a biological adsorbent and separated and filtered by a microporous filter membrane, so as to enrich the anti-inflammatory active ingredients, and obtain the functional tobacco seed oil with the anti-inflammatory active ingredients.
2. According to the invention, the yeast powder is adopted as a biological adsorbent for adsorption, and the primary oil of the tobacco seeds is separated and filtered by adopting the microporous filter membrane, so that heavy metals, salts, pigments, impurities and the like generated during cold pressing of the tobacco seeds can be effectively removed.
3. According to the invention, the semi-finished product of the tobacco seed oil is subjected to tertiary molecular distillation at different temperatures and pressures, the anti-inflammatory active ingredients of the tobacco seed oil can be effectively retained by strictly controlling the distillation temperature, the impurity ingredients in the tobacco seed oil can be separated again, and the high-purity functional tobacco seed oil with the anti-inflammatory active ingredients is obtained by tertiary molecular distillation.
4. The anti-inflammatory activity of the tobacco seed oil is comprehensively evaluated, so that the tobacco seed oil is determined to have anti-inflammatory active ingredients; the tobacco seed oil qualified by the evaluation of anti-inflammatory activity is added into vaseline to be used as anti-inflammatory cream, or added into beeswax to be used as lipstick for cheilitis, or directly used as anti-inflammatory essential oil to exert the anti-inflammatory activity 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 the cytotoxicity assay of aromatic tobacco Baoshan No. 4 tobacco seed oil in example 1 of the present invention;
FIG. 2 is a graph showing the measurement of the content of Nitric Oxide (NO), tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), and interleukin 6(IL-6) in LPS-induced RAW264.7 cells by aromatic tobacco Baoshan No. 4 tobacco seed oil and 10. mu.M Dexamethasone (DXM) in example 1 of the present invention;
in the figure, K: blank control group; m: a LPS group; y: positive group (dexamethasone)#P<0.05vs K group, P<0.05、**P<0.01vs group M;
FIG. 3 is the cytotoxicity assay of wild tobacco Rustica tobacco seed oil in example 2 of the present invention;
FIG. 4 is a graph showing the measurement of the content of Nitric Oxide (NO), tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), and interleukin 6(IL-6) in LPS-induced RAW264.7 cells by wild tobacco Rustica tobacco seed oil and 10. mu.M Dexamethasone (DXM) in example 2 of the present invention;
in the figure, K: blank control group; m: a LPS group; y: positive group (dexamethasone)#P<0.05vs K group, P<0.05、**P<0.01vs group M;
FIG. 5 is a cytotoxicity assay of cured tobacco NC89 tobacco seed oil in example 3 of the present invention;
FIG. 6 is a graph showing the content of nitrogen monoxide (NO), tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), and interleukin 6(IL-6) in LPS-induced RAW264.7 cells measured by flue-cured tobacco NC89 tobacco seed oil and 10. mu.M Dexamethasone (DXM) in example 3 of the present invention;
in the figure, K: blank control group; m: a LPS group; y: positive group (dexamethasone)#P<0.05vs K group, P<0.05、**P<0.01vs group M.
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
In this example, Baoshan No. 4 in aromatic tobacco was used as an example.
1.1A method for preparing tobacco seed oil with anti-inflammatory active ingredients comprises the following steps:
(1) selecting raw materials: collecting mature tobacco capsule No. 4 from Baoshan, and airing until the tobacco capsule No. 4 from Baoshan is dry; 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) crushing the dried plump Baoshan No. 4 tobacco seeds to 60 meshes, and performing cold pressing at the temperature of below 70 ℃ to obtain cold-pressed tobacco seed primary oil;
(3) centrifuging the cold-pressed tobacco seed primary oil at 7500r/min, and removing solid phase substances to obtain tobacco seed primary oil;
(4) adding yeast powder biological adsorbent into the tobacco seed primary oil, wherein the mass ratio of the yeast powder to the tobacco seed primary oil is 1:20, and placing the tobacco seed primary oil in a constant-temperature shaking table for oscillation adsorption reaction for 7 hours;
(5) after the reaction in the step (4) is finished, separating and filtering the mixture by using a 0.32 mu m microporous filter membrane to remove the yeast powder biological adsorbent to obtain a semi-finished product of the tobacco seed oil;
(6) putting the semi-finished product of the 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 95 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 105 ℃ 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 125 ℃ under the condition of 1Pa, and condensing to obtain tobacco seed oil L3 with a third fraction as a third-stage light component;
(7) and combining the tobacco seed oil L1, the tobacco seed oil L2 and the tobacco seed oil L3 to obtain the Baoshan No. 4 tobacco seed oil with the anti-inflammatory active ingredient.
1.2 carrying out GC-MS detection on the Baoshan No. 4 tobacco seed oil with the anti-inflammatory active ingredients, and determining the content of fatty acid and the content of volatile ingredients in the tobacco seed oil.
1.2.1 method for determining fatty acid content: and (3) extracting and enriching a No. 4 Baoshan tobacco seed oil sample 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 ℃. 3mL of Baoshan No. 4 tobacco seed oil with anti-inflammatory active ingredients is accurately weighed, added into a 20mL headspace bottle, immediately sealed and extracted for 20min at 40 ℃. 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 baoshan No. 4 tobacco seed oil with anti-inflammatory active ingredients are shown in table 1.
Figure BDA0003519531120000051
Figure BDA0003519531120000061
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 the Baoshan No. 4 tobacco seed oil with the anti-inflammatory active ingredients are shown in Table 2.
Figure BDA0003519531120000062
TABLE 2
1.3 determining the content influence of Baoshan No. 4 tobacco seed oil on NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in LPS-induced RAW264.7 cells.
1.3.1 determining cytotoxicity of Baoshan No. 4 tobacco seed oil, and obtaining specific concentration of the tobacco seed oil through the result of determining the cytotoxicity of Baoshan No. 4 tobacco seed oil to carry out a cell anti-inflammatory experiment.
The specific determination method comprises the following steps:
(1) cell digestion: RAW264.7 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 RAW264.7 cells in logarithmic growth phase to digestion and centrifugation according to the digestion method in (1) to obtain RAW264.7 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 to continue the culture. After the cells are treated for 4 hours, the supernatant is sucked away, 200 mu L of DMSO is added again, the crystal violet is fully dissolved by shaking in the dark, the 490nm light absorption value is measured by using an enzyme-linked immunosorbent assay, and the cell survival rate is calculated.
Cell viability (%) - (OD)Sample set-ODBlank group)/(ODControl group-ODBlank group)×100％。
The results of measuring the viability of RAW264.7 cells by using different concentrations of baoshan No. 4 tobacco seed oil are shown in fig. 1.
As can be seen from FIG. 1, the Baoshan No. 4 tobacco seed oil with different concentrations has small influence on the survival rate of RAW264.7 cells, wherein the Baoshan No. 4 tobacco seed oil with the concentration of 25-100 μ g/ml has no obvious cytotoxicity on the RAW264.7 cells. Therefore, 25, 50 and 100 mu g/ml are selected as the concentrations of Baoshan No. 4 tobacco seed oil to carry out the cell anti-inflammatory experiment.
1.3.2 measurement of NO inflammatory mediator content, TNF-alpha inflammatory factor content, IL-1 beta inflammatory factor content and IL-6 inflammatory factor content in LPS-induced RAW264.7 cells by Baoshan No. 4 tobacco seed oil
And (3) determining the content of NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in the LPS-induced RAW264.7 cells by using dexamethasone as a positive control and using the dexamethasone and the Baoshan No. 4 tobacco seed oil.
RAW264.7 cells were cultured according to the method of 1.3.1 at 1.0X 105Carrying out plate receiving per mL; after the cells are completely attached to the wall and grow, removing the culture medium, respectively adding 25, 50 and 100 mu g/ml Baoshan No. 4 tobacco seed oil and 10 mu M Dexamethasone (DXM) to treat RAW264.7 cells for 4h (replacing the culture medium for blank and model holes), and removing the supernatant after the treatment is finished; after the cells are washed by PBS, 1 mu g/mL of LPS is added again to continue culturing for 20h, and lysate is collected after the cells are lysed; and measuring the contents of Nitric Oxide (NO), tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta) and interleukin 6(IL-6) in the cells according to the kit specification.
25. The measurement results of 50, 100 μ g/ml concentrations of baoshan No. 4 tobacco seed oil and 10 μ M Dexamethasone (DXM) on the contents of Nitric Oxide (NO), tumor necrosis factor α (TNF- α), interleukin 1 β (IL-1 β), and interleukin 6(IL-6) in LPS-induced RAW264.7 cells are shown in fig. 2, where K: blank control group; m: a LPS group; y: positive group (dexamethasone)#P<0.05vs K group, P<0.05、**P<0.01vs group M.
As can be seen from the test results of fig. 2: LPS significantly induced the production of inflammatory mediators (NO) as well as inflammatory factors (TNF-alpha, IL-1 beta, IL-6) in RAW264.7 cells (P < 0.05). The Baoshan No. 4 tobacco seed oil can obviously inhibit the production (P <0.05, P <0.01) of inflammatory mediators (NO) and inflammatory factors (TNF-alpha, IL-1 beta and IL-6) in RAW264.7 cells in a dose-dependent mode, wherein the Baoshan No. 4 tobacco seed oil with 100 mu g/ml has the best inhibition effect which is equivalent to the inhibition effect of a positive group (dexamethasone), and can play a good role in resisting cell inflammation.
1.4 application of Baoshan No. 4 tobacco seed oil with anti-inflammatory active ingredients
And (3) determining the influence of the fatty acid content and the volatile component content of the Baoshan No. 4 tobacco seed oil determined in the step 1.2 and the Baoshan No. 4 tobacco seed oil in the step 1.3 on the content of NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in the RAW264.7 cells induced by LPS, so that the Baoshan No. 4 tobacco seed oil has stronger anti-inflammatory activity. The Baoshan No. 4 tobacco seed oil can be added into vaseline to serve as an anti-inflammatory cream, or added into beeswax to serve as a cheilitis lipstick, or directly serves as an anti-inflammatory essential oil, so that the Baoshan No. 4 tobacco seed oil has good anti-inflammatory activity.
Example 2
This example is an example of a Rustica variety in wild tobacco.
2.1A method for preparing tobacco seed oil with anti-inflammatory 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) crushing the dried plump Rustica tobacco seeds to the granularity of 65 meshes, and performing cold pressing at the temperature of below 70 ℃ to obtain cold pressed tobacco seed primary oil;
(3) centrifuging the cold-pressed tobacco seed primary oil at 7500r/min, and removing solid phase substances to obtain tobacco seed primary oil;
(4) adding yeast powder biological adsorbent into the tobacco seed primary oil, wherein the mass ratio of the yeast powder to the tobacco seed primary oil is 1:20, and placing the tobacco seed primary oil in a constant-temperature shaking table for oscillation adsorption reaction for 6 hours;
(5) after the reaction in the step (4) is finished, separating and filtering the mixture by using a 0.32 mu m microporous filter membrane to remove the yeast powder biological adsorbent to obtain a semi-finished product of the tobacco seed oil;
(6) putting the semi-finished product of the 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 90 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;
(7) and combining the tobacco seed oil L1, the tobacco seed oil L2 and the tobacco seed oil L3 to obtain the Rustica tobacco seed oil with the anti-inflammatory active ingredients.
2.2 carrying out GC-MS detection on the Rustica tobacco seed oil with the anti-inflammatory active ingredients, and determining the content of fatty acid and the content of volatile ingredients in the tobacco seed oil.
The method of measuring the fatty acid content and the method of measuring the volatile component content were the same as those in example 1.
The main fatty acid components of Rustica tobacco seed oil with anti-inflammatory active ingredient are shown in table 3.
Figure BDA0003519531120000091
TABLE 3
The volatile main components and the proportion of the Rustica tobacco seed oil with the anti-inflammatory active ingredients are shown in table 4.
Figure BDA0003519531120000101
TABLE 4
2.3 content of NO inflammatory mediator, TNF-alpha inflammatory factor, IL-1 beta inflammatory factor and IL-6 inflammatory factor in LPS-induced RAW264.7 cells by Rustica tobacco seed oil
2.3.1 performing cytotoxicity determination on the Rustica tobacco seed oil, and acquiring the specific concentration of the tobacco seed oil according to the cytotoxicity determination result of the Rustica tobacco seed oil to perform a cell anti-inflammatory experiment; the specific measurement method was the same as in example 1.
The results of the determination of the viability of RAW264.7 cells by different concentrations of Rustica tobacco seed oil are shown in FIG. 3.
As can be seen from FIG. 3, 25-100. mu.g/ml of Rustica tobacco seed oil had no significant cytotoxicity to RAW264.7 cells. Therefore, 25, 50 and 100 mu g/ml are selected as the concentrations of the Rustica tobacco seed oil to carry out the anti-inflammatory experiments on the cells.
2.3.2 the content of NO inflammatory mediators, TNF-alpha inflammatory factor, IL-1 beta inflammatory factor and IL-6 inflammatory factor in the LPS-induced RAW264.7 cells is measured by the Rustica tobacco seed oil, and the specific measurement method is the same as that in the example 1.
Dexamethasone is used as a positive control, the content of NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in LPS induced RAW264.7 cells by the dexamethasone and the Rustica tobacco seed oil is determined, and the detection result is shown in figure 4. In fig. 4, K: blank control group; m: a LPS group; y: positive group (dexamethasone), lower case letters indicate differential significance between treatments (P < 0.05).
As can be seen from FIG. 4, LPS significantly induced the production of inflammatory mediators (NO) and inflammatory factors (TNF- α, IL-1 β, IL-6) in RAW264.7 cells, and Rustica tobacco seed oil significantly inhibited the production of inflammatory mediators (NO) and inflammatory factors (TNF- α, IL-1 β, IL-6) in RAW264.7 cells in a dose-dependent manner. Compared with the inhibition effect of a positive group (dexamethasone), the Rustica tobacco seed oil can play a good role in cell anti-inflammation under a certain concentration.
2.4 application of Rustica tobacco seed oil with anti-inflammatory active ingredient
And (3) determining the influence of the content of fatty acid and volatile components of the Rustica tobacco seed oil determined in the step 2.2 and the content of NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in the RAW264.7 cells induced by LPS by the Rustica tobacco seed oil determined in the step 2.3, so that the Rustica tobacco seed oil has stronger anti-inflammatory activity. The Rustica tobacco seed oil can be added into vaseline as antiinflammatory cream, or added into beeswax as lipstick for cheilitis, or directly used as antiinflammatory essential oil, so that it has good antiinflammatory activity.
Example 3
The embodiment takes the NC89 variety in the flue-cured tobacco as an example.
3.1A method for preparing tobacco seed oil with anti-inflammatory 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 9% for later use;
(2) crushing the dried plump NC89 tobacco seeds to 70 meshes, and performing cold pressing at the temperature of below 70 ℃ to obtain cold pressed tobacco seed primary oil;
(3) centrifuging the cold-pressed tobacco seed primary oil at 7500r/min, and removing solid phase substances to obtain tobacco seed primary oil;
(4) adding yeast powder biological adsorbent into the tobacco seed primary oil, wherein the mass ratio of the yeast powder to the tobacco seed primary oil is 1:20, and placing the tobacco seed primary oil in a constant-temperature shaking table for oscillation adsorption reaction for 8 hours;
(5) after the reaction in the step (4) is finished, separating and filtering the mixture by using a 0.32 mu m microporous filter membrane to remove the yeast powder biological adsorbent to obtain a semi-finished product of the tobacco seed oil;
(6) putting the semi-finished product of the 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 100 deg.C under 100Pa to obtain first fraction of oleum Nicotianae L1 as first-stage light component; continuing performing secondary molecular distillation on the first distillation residue at 110 ℃ 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 130 ℃ under the condition of 1Pa, and condensing to obtain tobacco seed oil L3 with a third fraction as a third-stage light component;
(7) and 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 the anti-inflammatory active ingredients.
3.2 carrying out GC-MS detection on NC89 tobacco seed oil with the anti-inflammatory active ingredients, and determining the content of fatty acid and the content of volatile ingredients in the tobacco seed oil.
The method of measuring the fatty acid content and the method of measuring the volatile component content were the same as those in example 1.
The main fatty acid components of NC89 tobacco seed oil with anti-inflammatory active ingredients are shown in table 5.
Figure BDA0003519531120000111
Figure BDA0003519531120000121
TABLE 5
The volatile main components and the proportion of NC89 tobacco seed oil with anti-inflammatory active ingredients are shown in table 6.
Figure BDA0003519531120000122
TABLE 6
3.3 NC89 content of tobacco seed oil in LPS-induced RAW264.7 cells NO inflammatory mediator, TNF-alpha inflammatory factor, IL-1 beta inflammatory factor and IL-6 inflammatory factor
3.3.1 measuring the cytotoxicity of the NC89 tobacco seed oil, and obtaining the specific concentration of the tobacco seed oil according to the result of measuring the cytotoxicity of the NC89 tobacco seed oil to carry out a cell anti-inflammatory experiment; the specific measurement method was the same as in example 1.
The results of the measurement of the cell viability of RAW264.7 by the varying concentrations of NC89 tobacco seed oil are shown in fig. 5.
As can be seen from FIG. 5, NC89 smoke seed oil at 25-100. mu.g/ml has no significant cytotoxicity to RAW264.7 cells. Therefore, 25, 50 and 100 mu g/ml are selected as the concentration of NC89 tobacco seed oil to carry out the cell anti-inflammatory experiment.
3.3.2NC89 tobacco seed oil for measuring the content of NO inflammatory mediator, the content of TNF-alpha inflammatory factor, the content of IL-1 beta inflammatory factor and the content of IL-6 inflammatory factor in the LPS-induced RAW264.7 cells, the specific measuring method is the same as that in the embodiment 1.
The content of NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in LPS-induced RAW264.7 cells by dexamethasone and NC89 tobacco seed oil is determined by taking dexamethasone as a positive control, and the determination result is shown in figure 6, wherein K: blank control group; m: a LPS group; y: positive group (dexamethasone)#P<0.05vs K group, P<0.05、**P<0.01vs group M.
As can be seen from FIG. 6, LPS significantly induced the production of inflammatory mediators (NO) and inflammatory factors (TNF- α, IL-1 β, IL-6) in RAW264.7 cells, and NC89 smoke seed oil significantly inhibited the production of inflammatory mediators (NO) and inflammatory factors (TNF- α, IL-1 β, IL-6) in RAW264.7 cells in a dose-dependent manner. Compared with the inhibition effect of a positive group (dexamethasone), the NC89 tobacco seed oil can play a good cell anti-inflammatory role under a certain concentration.
3.4 application of NC89 tobacco seed oil with anti-inflammatory active ingredient
And (3) combining the content of fatty acid and the content of volatile components of NC89 tobacco seed oil measured in the step 3.2 and measuring the influence of NC89 tobacco seed oil in the step 3.3 on the content of NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in the RAW264.7 cells induced by LPS, the NC89 tobacco seed oil has stronger anti-inflammatory activity. NC89 oleum Nicotianae can be added into vaseline as antiinflammatory cream, or added into Cera flava as lipstick, or directly used as antiinflammatory essential oil to make it have good antiinflammatory activity.
Example 4
In this example, Baoshan No. 4 in aromatic tobacco was used as an example.
4.1A method for preparing tobacco seed oil with anti-inflammatory 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 8% for later use;
(2) crushing the dried plump Baoshan No. 4 tobacco seeds to the granularity of 65 meshes, and performing cold pressing at the temperature of below 70 ℃ to obtain cold-pressed tobacco seed primary oil;
(3) centrifuging the cold-pressed tobacco seed primary oil at 7000r/min, and removing solid phase substances to obtain tobacco seed primary oil;
(4) adding yeast powder biological adsorbent into the tobacco seed primary oil, wherein the mass ratio of the yeast powder to the tobacco seed primary oil is 1:20, and placing the tobacco seed primary oil in a constant-temperature shaking table for oscillation adsorption reaction for 7 hours;
(5) after the reaction in the step (4) is finished, separating and filtering the mixture by using a 0.32 mu m microporous filter membrane to remove the yeast powder biological adsorbent to obtain a semi-finished product of the tobacco seed oil;
(6) putting the semi-finished product of the 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 90 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;
(7) and (3) combining the tobacco seed oil L1, the tobacco seed oil L2 and the tobacco seed oil L3 to obtain the Baoshan No. 4 tobacco seed oil with the anti-inflammatory active ingredient.
4.2 carrying out GC-MS detection on the Baoshan No. 4 tobacco seed oil with the anti-inflammatory active ingredients, and determining the content of fatty acid and the content of volatile ingredients in the tobacco seed oil.
4.2.1 determination method of fatty acid content: a headspace solid phase microextraction method (HS-SPME) is adopted to extract and enrich Baoshan No. 4 tobacco seed oil samples, and 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 ℃. 3mL of Baoshan No. 4 tobacco seed oil with anti-inflammatory active ingredients is accurately weighed, added into a 20mL headspace bottle, immediately sealed and extracted for 20min at 40 ℃. 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 baoshan No. 4 tobacco seed oil with anti-inflammatory active ingredients are shown in table 1.
Figure BDA0003519531120000141
Figure BDA0003519531120000151
TABLE 7
4.2.2 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 the Baoshan No. 4 tobacco seed oil with the anti-inflammatory active ingredients are shown in Table 2.
Figure BDA0003519531120000152
TABLE 8
4.3 determining the content influence of Baoshan No. 4 tobacco seed oil on NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in LPS-induced RAW264.7 cells.
4.3.1 determining cytotoxicity of Baoshan No. 4 tobacco seed oil, and obtaining specific concentration of the tobacco seed oil according to the result of determining the cytotoxicity of Baoshan No. 4 tobacco seed oil to carry out a cell anti-inflammatory experiment.
The specific determination method comprises the following steps:
(1) cell digestion: RAW264.7 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 RAW264.7 cells in logarithmic growth phase to digestion and centrifugation according to the digestion method in (1) to obtain RAW264.7 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 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 results of measuring the viability of RAW264.7 cells by using different concentrations of baoshan No. 4 tobacco seed oil are shown in fig. 1.
As can be seen from FIG. 1, the Baoshan No. 4 tobacco seed oil with different concentrations has small influence on the survival rate of RAW264.7 cells, wherein the Baoshan No. 4 tobacco seed oil with the concentration of 25-100 μ g/ml has no obvious cytotoxicity on the RAW264.7 cells. Therefore, 25, 50 and 100 mu g/ml are selected as the concentrations of Baoshan No. 4 tobacco seed oil to carry out the cell anti-inflammatory experiment.
4.3.2 measurement of NO inflammatory mediator content, TNF-alpha inflammatory factor content, IL-1 beta inflammatory factor content and IL-6 inflammatory factor content in LPS-induced RAW264.7 cells by Baoshan No. 4 tobacco seed oil
And (3) determining the content of NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in LPS-induced RAW264.7 cells by using dexamethasone as a positive control and by using the dexamethasone and the Baoshan No. 4 tobacco seed oil.
RAW264.7 cells were cultured according to the method of 1.3.1 at 1.0X 105Carrying out plate receiving per mL; after the cells are completely attached to the wall and grow, removing the culture medium, respectively adding 25, 50 and 100 mu g/ml Baoshan No. 4 tobacco seed oil and 10 mu M Dexamethasone (DXM) to treat RAW264.7 cells for 4h (replacing the culture medium for blank and model holes), and removing the supernatant after the treatment is finished; after the cells are washed by PBS, 1 mu g/mL of LPS is added again to continue culturing for 20h, and lysate is collected after the cells are lysed; and measuring the contents of Nitric Oxide (NO), tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta) and interleukin 6(IL-6) in the cells according to the kit specification.
The Baoshan No. 4 tobacco seed oil can obviously inhibit the production (P <0.05, P <0.01) of inflammatory mediators (NO) and inflammatory factors (TNF-alpha, IL-1 beta and IL-6) in RAW264.7 cells in a dose-dependent mode, wherein the Baoshan No. 4 tobacco seed oil with 100 mu g/ml has the best inhibition effect which is equivalent to the inhibition effect of a positive group (dexamethasone), and can play a good role in resisting cell inflammation.
4.4 application of Baoshan No. 4 tobacco seed oil with anti-inflammatory active ingredient
And the content of fatty acid and volatile components of the Baoshan No. 4 tobacco seed oil measured in the step 4.2 and the content influence of the Baoshan No. 4 tobacco seed oil in the step 4.3 on NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in the RAW264.7 cells induced by LPS are measured, so that the Baoshan No. 4 tobacco seed oil has strong anti-inflammatory activity. The Baoshan No. 4 tobacco seed oil can be added into vaseline to serve as an anti-inflammatory cream, or added into beeswax to serve as a cheilitis lipstick, or directly serves as an anti-inflammatory essential oil, so that the Baoshan No. 4 tobacco seed oil has good anti-inflammatory activity.
Example 5
In this example, Baoshan No. 4 in aromatic tobacco was used as an example.
5.1A method for preparing tobacco seed oil with anti-inflammatory active ingredient 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 tobacco seeds of Baoshan No. 4, cleaning the plump tobacco seeds, and drying until the moisture content is reduced to 10% for later use;
(2) crushing the dried plump Baoshan No. 4 tobacco seeds to 70 meshes, and performing cold pressing at the temperature of below 70 ℃ to obtain cold-pressed tobacco seed primary oil;
(3) centrifuging the cold-pressed tobacco seed primary oil at 8000r/min, and removing solid phase substances to obtain tobacco seed primary oil;
(4) adding yeast powder biological adsorbent into the tobacco seed primary oil, wherein the mass ratio of the yeast powder to the tobacco seed primary oil is 1:20, and placing the tobacco seed primary oil in a constant-temperature shaking table for oscillation adsorption reaction for 7 hours;
(5) after the reaction in the step (4) is finished, separating and filtering the mixture by using a 0.32 mu m microporous filter membrane to remove the yeast powder biological adsorbent to obtain a semi-finished product of the tobacco seed oil;
(6) putting the semi-finished product of the 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 100 deg.C under 100Pa to obtain first fraction of oleum Nicotianae L1 as first-stage light component; continuing performing secondary molecular distillation on the first distillation residue at 110 ℃ 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 130 ℃ and under the condition of 1Pa, and condensing to obtain tobacco seed oil L3 with a third fraction as a three-stage light component;
(7) and combining the tobacco seed oil L1, the tobacco seed oil L2 and the tobacco seed oil L3 to obtain the Baoshan No. 4 tobacco seed oil with the anti-inflammatory active ingredient.
5.2 carrying out GC-MS detection on the Baoshan No. 4 tobacco seed oil with the anti-inflammatory active ingredients, and determining 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 a No. 4 Baoshan tobacco seed oil sample 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 ℃. 3mL of Baoshan No. 4 tobacco seed oil with anti-inflammatory active ingredients is accurately weighed, added into a 20mL headspace bottle, immediately sealed and extracted for 20min at 40 ℃. 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 baoshan No. 4 tobacco seed oil with anti-inflammatory active ingredients are shown in table 1.
Figure BDA0003519531120000181
Figure BDA0003519531120000191
TABLE 9
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 the Baoshan No. 4 tobacco seed oil with the anti-inflammatory active ingredients are shown in Table 2.
Figure BDA0003519531120000192
Watch 10
5.3 determining the content influence of No. 4 tobacco seed oil on NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in the RAW264.7 cells induced by LPS.
5.3.1 determining cytotoxicity of Baoshan No. 4 tobacco seed oil, and obtaining specific concentration of the tobacco seed oil according to the result of determining the cytotoxicity of Baoshan No. 4 tobacco seed oil to carry out a cell anti-inflammatory experiment.
The specific determination method comprises the following steps:
(1) cell digestion: RAW264.7 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 RAW264.7 cells in logarithmic growth phase to digestion and centrifugation according to the digestion method in (1) to obtain RAW264.7 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 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％。
5.3.2 measurement of NO inflammatory mediator content, TNF-alpha inflammatory factor content, IL-1 beta inflammatory factor content and IL-6 inflammatory factor content in LPS-induced RAW264.7 cells by Baoshan No. 4 tobacco seed oil
And (3) determining the content of NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in the LPS-induced RAW264.7 cells by using dexamethasone as a positive control and using the dexamethasone and the Baoshan No. 4 tobacco seed oil.
RAW264.7 cells were cultured according to the method of 1.3.1 at 1.0X 105Carrying out plate receiving per mL; after the cells are completely attached to the wall and grow, removing the culture medium, respectively adding 25, 50 and 100 mu g/ml Baoshan No. 4 tobacco seed oil and 10 mu M Dexamethasone (DXM) to treat RAW264.7 cells for 4h (replacing the culture medium for blank and model holes), and removing the supernatant after the treatment is finished; after the cells are washed by PBS, 1 mu g/mL of LPS is added again to continue culturing for 20h, and lysate is collected after the cells are lysed; and measuring the contents of Nitric Oxide (NO), tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta) and interleukin 6(IL-6) in the cells according to the kit specification.
The Baoshan No. 4 tobacco seed oil can obviously inhibit the production (P <0.05, P <0.01) of inflammatory mediators (NO) and inflammatory factors (TNF-alpha, IL-1 beta and IL-6) in RAW264.7 cells in a dose-dependent mode, wherein the Baoshan No. 4 tobacco seed oil with 100 mu g/ml has the best inhibition effect which is equivalent to the inhibition effect of a positive group (dexamethasone), and can play a good role in resisting cell inflammation.
5.4 application of Baoshan No. 4 tobacco seed oil with anti-inflammatory active ingredient
And the content of fatty acid and volatile components of the Baoshan No. 4 tobacco seed oil measured in the step 5.2 and the content influence of the Baoshan No. 4 tobacco seed oil in the step 5.3 on NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in the RAW264.7 cells induced by LPS are measured, so that the Baoshan No. 4 tobacco seed oil has strong anti-inflammatory activity. The Baoshan No. 4 tobacco seed oil can be added into vaseline to serve as an anti-inflammatory cream, or added into beeswax to serve as a cheilitis lipstick, or directly serves as an anti-inflammatory essential oil, so that the Baoshan No. 4 tobacco seed oil has good anti-inflammatory activity.
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 (9)

1. A preparation method of tobacco seed oil with anti-inflammatory active ingredients is characterized by comprising 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) crushing the plump tobacco seeds dried in the step (1), and then performing cold pressing to obtain cold pressed tobacco seed primary oil;
(3) centrifuging the tobacco seed primary oil in the step (2) at 7000-8000r/min, and then discarding solid phase substances to obtain the tobacco seed primary oil;
(4) adding a biological adsorbent into the tobacco seed primary oil in the step (3), and placing the mixture in a constant-temperature shaking table for oscillation adsorption reaction for 6-8 hours;
(5) after the reaction in the step (4) 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;
(6) putting the semi-finished product of the tobacco seed oil in the step (5) into a molecular distillation instrument, carrying out first molecular distillation at 90-100 ℃ under the condition of 100Pa, and obtaining tobacco seed oil L1 with first fraction as a first-stage light component after condensation; continuing performing secondary molecular distillation on the primary distillation residue at the temperature of 100-110 ℃ and under the condition of 10Pa, and condensing to obtain a second fraction which is the tobacco seed oil L2 of the secondary light component; continuously carrying out three-stage molecular distillation on the second distillation residue at the temperature of 120-130 ℃ and under the condition of 1Pa, and condensing to obtain smoke seed oil L3 with a third fraction as a three-stage light component;
(7) and (4) combining the tobacco seed oil L1, the tobacco seed oil L2 and the tobacco seed oil L3 in the step (6) to obtain the tobacco seed oil with the anti-inflammatory active ingredients.
2. The method for preparing the tobacco seed oil with the anti-inflammatory active ingredient as claimed in claim 1, wherein the cold pressing in the step (2) is performed at 70 ℃ or below after the plump tobacco seeds are crushed to the particle size of 60-70 meshes.
3. The method for preparing the tobacco seed oil with the anti-inflammatory active ingredient in the claim 1, wherein the biological adsorbent in the step (4) is yeast powder, and the mass ratio of the yeast powder to the tobacco seed primary oil is 1: 20.
4. The method for preparing the tobacco seed oil with the anti-inflammatory active ingredient as claimed in claim 1, wherein the pore size of the microporous filter membrane in the step (5) is 0.32 μm.
5. The method for preparing the tobacco seed oil with the anti-inflammatory active ingredients in claim 1, wherein the feeding speed of the molecular distillation instrument in the step (6) is 50mL/h, and the rotating speed of a rotor is 120 r/min.
6. A method for evaluating the anti-inflammatory activity of tobacco seed oil is characterized by comprising the following steps: the method comprises the following steps:
s1: subjecting the tobacco seed oil with anti-inflammatory active ingredients of any one of claims 1-5 to GC-MS detection to detect the content of fatty acids and volatile components in the tobacco seed oil with anti-inflammatory active ingredients;
s2: measuring the content of NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in LPS-induced RAW264.7 cells by using 10 mu M dexamethasone as a positive control;
s3: selecting 25-100 mu g/ml of tobacco seed oil, and determining the content of NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in the RAW264.7 cells induced by LPS by the tobacco seed oil with different concentrations;
s4: comparing the content of NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in LPS-induced RAW264.7 cells by 10 mu M dexamethasone in the step S2 with the content of NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in LPS-induced RAW264.7 cells by tobacco seed oil with different corresponding concentrations in the step S3, and determining the anti-inflammatory activity capability of the tobacco seed oil;
s5: and (4) comprehensively evaluating the anti-inflammatory activity of the tobacco seed oil by combining the anti-inflammatory activity of the tobacco seed oil determined in the step S4, the fatty acid content and the volatile component content of the tobacco seed oil determined in the step S1.
7. The method for evaluating the anti-inflammatory activity of the tobacco seed oil according to claim 6, wherein the GC-MS detection conditions for the fatty acid content in the step S1 are as follows:
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, programmed temperature 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 the anti-inflammatory activity of tobacco seed oil according to claim 6, wherein the content of NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in LPS-induced RAW264.7 cells is measured by dexamethasone or tobacco seed oil in step S2 or step S3:
culturing RAW264.7 cells and inoculating the cells in a culture medium, and removing the culture medium after the RAW264.7 cells grow in a complete adherent manner; adding 25-100 μ g/ml tobacco seed oil and 10 μ M dexamethasone to treat RAW264.7 cells, removing supernatant after treatment, washing RAW264.7 cells with PBS, adding LPS again to continue culturing; after the culture is finished, collecting lysate, and determining the content of NO inflammatory mediators, TNF-alpha inflammatory factors, IL-1 beta inflammatory factors and IL-6 inflammatory factors in RAW264.7 cells according to the kit specification.
9. The application of the tobacco seed oil with the anti-inflammatory active ingredient is characterized in that the tobacco seed oil with the anti-inflammatory active ingredient is added into vaseline to be used as an anti-inflammatory cream after the anti-inflammatory activity is evaluated to be qualified, or added into beeswax to be used as a cheilitis lipstick, or directly used as an anti-inflammatory essential oil.
CN202210173537.2A 2022-02-24 2022-02-24 Preparation method and application of smoke seed oil with anti-inflammatory active ingredient Active CN114525167B (en)

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