CN110129057B - Fragrance Rhododendron extract and its extraction method and use - Google Patents
Fragrance Rhododendron extract and its extraction method and use Download PDFInfo
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- CN110129057B CN110129057B CN201910372308.1A CN201910372308A CN110129057B CN 110129057 B CN110129057 B CN 110129057B CN 201910372308 A CN201910372308 A CN 201910372308A CN 110129057 B CN110129057 B CN 110129057B
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- 238000000605 extraction Methods 0.000 title abstract description 32
- 239000003205 fragrance Substances 0.000 title 1
- 238000000034 method Methods 0.000 claims abstract description 32
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- 239000001149 (9Z,12Z)-octadeca-9,12-dienoate Substances 0.000 claims description 34
- WTTJVINHCBCLGX-UHFFFAOYSA-N (9trans,12cis)-methyl linoleate Natural products CCCCCC=CCC=CCCCCCCCC(=O)OC WTTJVINHCBCLGX-UHFFFAOYSA-N 0.000 claims description 18
- LNJCGNRKWOHFFV-UHFFFAOYSA-N 3-(2-hydroxyethylsulfanyl)propanenitrile Chemical compound OCCSCCC#N LNJCGNRKWOHFFV-UHFFFAOYSA-N 0.000 claims description 18
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- DVWSXZIHSUZZKJ-YSTUJMKBSA-N methyl linolenate Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(=O)OC DVWSXZIHSUZZKJ-YSTUJMKBSA-N 0.000 claims description 16
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- C09K15/00—Anti-oxidant compositions; Compositions inhibiting chemical change
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- 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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
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Abstract
本发明提供烈香杜鹃提取物及提取方法，将烈香杜鹃的叶枝分离，用超临界CO2萃取法分别提取得到杜鹃叶部位及枝部位的提取物(挥发油)，利用GC‑MS法分析鉴定了所得杜鹃叶和枝部位总挥发油的化合物组成，并对其抗氧化活性进行了测定分析。本发明有利于更加科学和高效地进行烈香杜鹃资源的综合利用。
The invention provides an extract of Rhododendron fragrans and an extraction method. The leaves and branches of Rhododendron fragrans are separated, and the extracts (volatile oils) of the leaves and branches of Rhododendron are extracted respectively by a supercritical CO 2 extraction method, and the GC-MS method is used for analysis and identification. The compound composition of the total volatile oil from leaves and branches of Rhododendron rhododendron was obtained, and its antioxidant activity was determined and analyzed. The invention is conducive to more scientific and efficient comprehensive utilization of the Rhododendron fragrans resources.
Description
Technical Field
The invention belongs to the field of plant extracts, and particularly relates to extraction of compounds in rhododendron anthopogonoides.
Background
Rhododendron anthopogonoides Maxim) is evergreen shrub of Rhododendron of Ericaceae, and is mainly produced in Gansu, Qinghai and northwest Sichuan. It is usually planted in mountain slope with elevation of 2900-. As recorded in Tibetan medicine, it uses flowers, leaves and twigs as medicinal parts, is bitter, astringent and cold in nature, and has the effects of clearing heat, diminishing inflammation, relieving cough and asthma, invigorating stomach, strengthening body constitution, resisting aging, etc. Modern pharmacological researches find that geranone, alpha-limonene, benzyl acetone and juniper camphor in volatile oil parts of the volatile oil have the effect of relieving cough, gamma-apiene, juniper camphor, rhododendrin, farrerone and limonene have the effect of eliminating phlegm, limonene, benzyl acetone, geranone, rhododendrin, gamma-apiene, juniper camphor and the like all have certain antibacterial effect, and the total volatile oil has the obvious effect of expanding blood vessels.
At present, the research on the total volatile oil of rhododendron anthopogonoides is mainly focused on leaf or flower parts of the rhododendron anthopogonoides, and almost no research report on the total volatile oil of the branch parts of the rhododendron anthopogonoides is found. The existing Bianxiangdu teaThe extraction of the compounds from Rhododendron employs steam distillation method. Although the method is simple, the yield is not high, and the extraction temperature is high, so that the extract components are easy to damage. Qianweiguang et al studied the use of supercritical CO by an orthogonal test method2The best process for extracting the total volatile oil from the rhododendron leaf part by an extraction method is adopted, but the chemical components of the extract and the corresponding effects of the compounds are not analyzed.
On the other hand, different regions and climatic conditions exist, the functional components with beneficial effects contained in rhododendron anthopogonoides are different, and the content of the beneficial components in different parts of rhododendron anthopogonoides is also different. Therefore, in the comprehensive utilization of rhododendron anthopogonoides, the extraction of compounds in rhododendron anthopogonoides and the analysis and identification of components are particularly important.
Disclosure of Invention
The invention aims to provide an extract of rhododendron anthopogonoides branches and leaves and an extraction method, which are convenient for the comprehensive development and utilization of rhododendron anthopogonoides.
The invention firstly separates leaves and branches of rhododendron anthopogonoides and uses supercritical CO2Extracting to obtain folium Rhododendri Simsii extract and branch extract, i.e. branch oil and leaf oil, first analyzing and identifying the composition of the volatile oil by GC-MS method, and determining and analyzing its antioxidant activity.
The invention provides a rhododendron anthopogonoides extract, which comprises the following components obtained by first identification: 5-37% of linoleic acid, 6-24% of linolenic acid and 1-16% of delta-cnaphinene.
Since the GC-MS detection of fatty acids requires the prior methyl esterification, the percentage of fatty acids (including saturated and unsaturated fatty acids) in the extract is calculated according to the mass percentage of the corresponding fatty acid methyl ester of the fatty acid in the claims and the specification of the invention.
Linoleic acid is a functional polyunsaturated fatty acid, an essential fatty acid for humans, essential for human and mammalian growth, and cannot be synthesized in the body, and must be taken from food. It has the effect of reducing serum cholesterol level, and has obvious curative effect on patients with high triglyceride content by taking a large amount of linoleic acid. The Chinese pharmacopoeia still adopts ethyl linoleate pills and drops as the medicine for preventing and treating hypertension, atherosclerosis and coronary heart disease. Linoleic acid helps to reduce serum cholesterol and inhibit arterial thrombosis, thus having good effects in preventing cardiovascular diseases such as atherosclerosis and myocardial infarction. Furthermore, linoleic acid is also a precursor of omega-6 long chain polyunsaturated fatty acids, especially gamma-linolenic acid and arachidonic acid. Linolenic acid is also not made by the human body and must be ingested from food, and has a series of health-care effects: reducing blood lipid and cholesterol; reducing blood pressure; anti-cancer effect; improving cardiovascular and cerebrovascular diseases; improving cranial nerve function, etc. Therefore, the rhododendron anthopogonoides extract provided by the invention provides a rich, cheap and economic source for linolenic acid and linoleic acid, and can be used as an additive component of a health-care product for supplementing the linolenic acid and the linoleic acid; or further processing into health product; or used as the extraction raw materials of linoleic acid and linolenic acid as functional components in the medicine development and used as auxiliary materials in the medicine preparation; or used as chemical raw material and additive, etc., and can be used as synthetic intermediate of omega-6 long-chain polyunsaturated fatty acid, especially gamma-linolenic acid and arachidonic acid.
Further, the extract is branch oil extracted from rhododendron anthopogonoides branches or leaf oil extracted from rhododendron anthopogonoides leaves;
the branch oil contains, by mass, 5.5-6% of linoleic acid, 6-6.5% of linolenic acid, 15-15.5% of delta-cnidium and 6-7% of 4-phenyl-2-butanone; further, the branch oil also comprises 0.1-0.6% of beta-maleic acid, 0.1-1% of cadinene, 0.5-2% of 3, 9-cadiene, 0.5-1.5% of 4(14),7(11) -eudesmadiene, 1-2% of eicosane, 1-2% of tetracosane, 1-2% of 2-heptadecanone, 1-2% of hexacosane, 1.5-2.5% of 2-nonadecanone, 3.8-5% of nonacosane and 1.5-2.5% of Turkish balsone in percentage by mass;
the leaf oil contains, by mass, 35-37% of methyl linoleate, 22-24% of methyl linolenate, 1-2% of delta-cnidiene and 0.1-1.0% of 4-phenyl-2-butanone; further, the leaf oil also comprises 0.05 to 0.4 percent of methyl dodecanoate, 0.1 to 0.5 percent of 1(10) 6.8-cadotriene, 0.1 to 0.5 percent of methyl heptadecanoate and 0.0 to 0.6 percent of methyl oleate in percentage by mass.
Furthermore, the rhododendron branch oil also comprises the following components in percentage by mass: 1.5-2.5% of alpha-maleic elemene, 6-9% of gamma-eucalyptol, 1-2% of methyl myristate, 2.5-3.5% of methyl palmitate, 2-3% of methyl stearate, 2-3% of methyl 11 cis-octadecenoate, 12.5-14.5% of heptacosane, 2.6-3.8% of methyl docosanate and 6-7% of methyl tetracosanate;
the leaf oil also comprises the following components in percentage by mass: 0.1-1% of methyl myristate, 7.5-9.5% of methyl palmitate, 1.5-2.5% of methyl stearate, 8.5-10.5% of methyl 11 cis-octadecenoate, 2.5-3.5% of methyl behenate and 7-8% of methyl lignoceric acid.
The invention provides edible oil which contains rhododendron leaf oil, wherein the rhododendron leaf oil comprises the following components in percentage by mass: 35 to 37 percent of methyl linoleate, 22 to 24 percent of methyl linolenate, 1 to 2 percent of delta-cnaphenylene, 0.1 to 0.4 percent of methyl dodecanoate, 0.1 to 0.4 percent of 1(10) 6.8-junipentane, 0.1 to 0.4 percent of methyl heptadecanoate, 0.1 to 0.4 percent of methyl oleate,
further, the edible oil also comprises 0.1-0.8% of gamma-eucalyptol, 8-9% of methyl palmitate and 9-10% of methyl 11 cis-octadecenoate.
The rhododendron anthopogonoides extract provided by the invention contains a large amount of linoleic acid (36.61%) and linolenic acid (23.84%). Linoleic Acid (LA) is the first recognized one of the functional polyunsaturated fatty acids (PUFAs), the Essential Fatty Acid (EFA), essential for human and mammalian growth, but is not synthesized in the body and must be ingested from food. It has the effect of reducing serum cholesterol level, and has obvious curative effect on patients with high triglyceride content by taking a large amount of linoleic acid. The Chinese pharmacopoeia still adopts ethyl linoleate pills and drops as the medicine for preventing and treating hypertension, atherosclerosis and coronary heart disease. Linoleic acid helps to reduce serum cholesterol and inhibit arterial thrombosis, thus having good effects in preventing cardiovascular diseases such as atherosclerosis and myocardial infarction. Furthermore, linoleic acid is also a precursor of omega-6 long chain polyunsaturated fatty acids, especially gamma-linolenic acid and arachidonic acid. Linolenic acid is also not made by the human body and must be ingested from food, and has a series of health-care effects: reducing blood lipid and cholesterol; reducing blood pressure; anti-cancer effect; improving cardiovascular and cerebrovascular diseases; improving cranial nerve function, etc. Based on the above, the rhododendron leaf oil can be considered to be developed into an edible oil product or be added into the existing edible oil as an additive component
The invention provides application of the rhododendron anthopogonoides extract in preparing relevant antioxidant products.
According to experimental verification, the rhododendron anthopogonoides extract provided by the invention has strong oxidation resistance, and can be added into products with oxidation resistance to play the oxidation resistance. The antioxidant related product can be a skin care product, a health care product and the like.
According to the report of the literature, 4-phenyl-2-butanone is a main chemical component in rhododendron oil which plays a pharmacodynamic activity role and has the effects of relieving cough and asthma; effects on respiration, blood pressure; the effect of prolonging sleep; slowing heart rate and reducing the effects of cardiac contractility; has obvious relaxation effect on guinea pig isolated tracheal smooth muscle and can resist contraction effect caused by histamine. The experimental research of the invention finds that the content of the 4-phenyl-2-butanone in the leaves is only 0.78 percent, and the content of the 4-phenyl-2-butanone in the branches is as high as 6.63 percent, which indicates that the rhododendron branch oil has higher development and utilization value of the 4-phenyl-2-butanone. Meanwhile, volatile oil in the twig is extracted by a traditional steam distillation method, the content of the obtained 4-phenyl-2-butanone is only 1.584 percent by determination, and the content of the 4-phenyl-2-butanone in the twig oil obtained by the method under the condition of a supercritical carbon dioxide extraction method is as high as 6.63 percent. In addition, the invention finds that the content of delta-cnaphinene in rhododendron anthopogonoides branch extract is as high as 15.15%, the content of delta-cnaphinene in leaf extract is only 1.61%, and delta-cnaphinene can be preferentially extracted from branches when the delta-cnaphinene needs to be extracted.
Therefore, the invention provides a method for extracting 4-phenyl-2-butanone and/or delta-cnidiene from rhododendron anthopogonoides, which takes rhododendron anthopogonoides branches as extraction raw materials, adopts a supercritical carbon dioxide extraction method, and extracts at the extraction temperature of 48-52 ℃, the separation temperature of 35-39 ℃ and the extraction pressure of 26-30 Mpa.
Further, it is preferable to conduct the extraction at an extraction temperature of 50 ℃ and a separation temperature of 37 ℃ under an extraction pressure of 28 MPa.
According to experimental research, the content difference of the methyl linoleate and the methyl linolenate in the branches and leaves and the leaves of rhododendron anthopogonoides is large, and the content of the methyl linoleate and the methyl linolenate in the leaves is respectively 36.61% and 23.84%. Therefore, the invention provides a method for extracting methyl linoleate and/or methyl linolenate from rhododendron anthopogonoides, which takes rhododendron anthopogonoides leaves as an extraction raw material and adopts a supercritical carbon dioxide extraction method to extract at the extraction temperature of 50 ℃, the separation temperature of 37 ℃ and the extraction pressure of 28 Mpa.
The invention provides a method for identifying rhododendron anthopogonoide branch oil and leaf oil, which is used for detecting whether an rhododendron anthopogonoide extract simultaneously contains the following marker compounds: methyl linoleate, methyl linolenate, delta-cnidium lactone, 4-phenyl-2-butanone, cadinene, 3, 9-cadiene, 4(14),7(11) -eucalyptene, 2-heptadecanone, 2-nonadecanone, nonacosane and cumarone, and if the compounds are contained, judging the rhododendron oil; or detecting whether the rhododendron anthopogonoides extract simultaneously contains the following marker compounds: methyl linoleate, methyl linolenate, delta-cnidium lactone, 4-phenyl-2-butanone, cadinene, 3, 9-cadiene, 4(14), (7), (11) -eucalyptene, 2-heptadecanone, 2-nonadecanone and cumarone, and if the compounds are contained at the same time, judging that the rhododendron oil is contained
Or detecting whether the rhododendron anthopogonoides extract simultaneously contains the following marker compounds: methyl linoleate, methyl linolenate, delta-cnidium lactone, 4-phenyl-2-butanone, methyl dodecanoate, 1(10)6, 8-cadotriene, methyl heptadecanoate and methyl oleate, and if the methyl linoleate, the methyl linolenate, the delta-cnidium lactone, the methyl 4-phenyl-2-butanone, the methyl dodecanoate and the methyl oleate are contained simultaneously, the rhododendron leaf oil is judged.
The 4-phenyl-2-butanone serving as a main chemical component playing a pharmacodynamic activity role in the rhododendron oil has the effects of relieving cough and asthma and has the influence on respiration and blood pressure. When the rhododendron branch oil and the rhododendron leaf oil are identified, the existence of the compound can be used as a standard for judging whether the identified product is the rhododendron volatile oil with corresponding medicinal value. The detection and identification method provided by the invention preferably adopts a gas chromatography-mass spectrometry combined method to detect the compound, and comprises the following conditions:
gas chromatography conditions: the sample inlet temperature is 240 ℃, and the column temperature adopts programmed temperature rise: maintaining the initial temperature at 40 deg.C for 5min, and increasing to 250 deg.C at 4 deg.C/min for 15 min; (ii) a A detector: MSD, detector temperature 230 ℃; carrier gas N2The flow rate is 1.0 mL/min; the split-flow and sample injection split ratio is 10: 1.
Mass spectrum conditions: the interface temperature is 280 ℃, the ion source temperature is 230 ℃, the electron bombardment ion source (EI) is 70eV, and the mass scanning range is 30-600 m/z.
Further, the following detection conditions are also included:
chromatographic conditions are as follows: column DB-5MS column (30m × 0.25mm × 0.25 um); sample injector: PSS; carrier gas: high purity helium gas; the column flow rate is 1.0 mL/min; sample introduction amount: 1 ul;
mass spectrum conditions: the instrument comprises the following steps: thermo Trace1300/8000Evo GC/MS/MS gas phase tandem mass spectrometer.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention provides a new rhododendron anthopogonoide extract, wherein the contents of methyl linoleate and methyl linolenate are respectively 36.61% and 23.84%, and the invention provides a new product with high oxidation resistance, which can be used as an oxidation resistant component to be added into products with oxidation resistance.
2. The invention respectively detects the chemical components of the branch part and the leaf part of the rhododendron anthopogonoides, finds different components in the branch and the leaf and different contents of the same component, provides a direction for more scientifically and efficiently utilizing the branch part and the leaf part of the rhododendron anthopogonoide, and simultaneously provides an effective method for identifying the extraction part of the rhododendron anthopogonoide extract.
3. The invention adopts the supercritical carbon dioxide extraction method for extraction, has low extraction temperature, is not easy to cause the damage of the extract components and has high extraction efficiency. The supercritical carbon dioxide extraction method shows that the oil yield of the rhododendron leaf oil (4.67 percent) is higher than that of the rhododendron branch oil (2.81 percent), and the oil yield extracted by the method is obviously higher than that extracted by the existing steam distillation method (1.5 percent), so that the method is particularly suitable for extracting the total volatile oil of the rhododendron branches and leaves.
Drawings
FIG. 1 is a total ion flow chromatogram of rhododendron oil;
FIG. 2 is a total ion flow chromatogram of rhododendron branch oil;
FIG. 3 is an ion fragment diagram of 4-phenyl-2-butanone;
FIG. 4 is an ion fragmentation pattern for methyl linoleate;
FIG. 5 is an ion fragmentation pattern for methyl linoleate;
FIG. 6 is a fragmentation pattern for methyl oleate ions;
FIG. 7 is an ion fragmentation pattern for methyl palmitate;
FIG. 8 is an ion fragmentation pattern for delta-cnidiacene;
FIG. 9 is an ion fragmentation pattern of gamma-eucalyptol;
FIG. 10 shows the azalea leaf oil clearance at 30min in DPPH clearance experiments;
FIG. 11 shows the azalea leaf oil clearance at 60min in the antioxidant activity test in DPPH removal experiments;
FIG. 12 is a graph of Rhododendron oil clearance at 30min in the antioxidant activity test;
FIG. 13 shows the Rhododendron oil clearance at 60min in the antioxidant activity test;
FIG. 14 ABTS method Rhododendron oil antioxidant capacity;
FIG. 15 shows the antioxidant activity of Rhododendron oil by ABTS method;
FIG. 16 FRAP method rhododendron leaf oil antioxidant capacity;
FIG. 17 oxidation resistance of Rhododendron oil by FRAP method.
Detailed Description
The invention is further illustrated by the following specific examples and experimental studies.
Example 1 extraction and composition analysis of Rhododendron anthopogonoides Branch oil and leaf oil
1. Instruments and reagents
The instrument comprises the following steps: HA221-40-12 model supercritical fluid extractor; 1L device. The analysis instrument is Thermo Trace13008000Evo GC/MS/MS gas phase tandem mass spectrometer, the detector model is MSD; the chromatographic column is Agilent DB-5MS UI (30m × 0.25mm × 0.25 um); epoch2Microplate spectrophotometer, beton instruments ltd (BioTek), usa.
Reagent: CO 22The gas is food grade (purity is more than or equal to 99%, Beijing medical gas company), kit FRAP, ABTS (Nanjing institute of bioengineering), DPPH.
Sample source: overground branches and leaves (branches and leaves in the sprouting period of tender leaves) of Rhododendron anthoporoides are collected from the province of Qinghai province, and are identified as Rhododendron anthopogonoides Maxim by Luzhong researchers of northwest institute of plateau biology of Chinese academy of sciences, overground parts are manually cut and separated into Rhododendron branches and Rhododendron leaves, impurities are removed, the branches and leaves are dried in the shade and are crushed for later use.
2. Extraction of rhododendron branch oil and rhododendron leaf oil
Subjecting branches and leaves of Rhododendron to supercritical CO respectively2Extracting by an extraction method.
Extraction conditions are as follows: the extraction temperature is 50 ℃, the separation temperature is 37 ℃, and the extraction pressure is 28 Mpa.
The extraction rate is as follows: the oil yield of the rhododendron leaves is 4.67 percent, and the oil yield of the rhododendron branches is 2.81 percent.
3. Determination of chemical composition of volatile oil
Gas chromatography conditions: sample injector: PSS, split-flow sample injection, the split-flow ratio is 10: 1; sample injector temperature: 240 ℃; carrier gas: high purity helium gas; the column flow rate is 1.0 mL/min; temperature programming: initial temperature of 40 deg.C (keeping for 5min), and raising temperature of 4 deg.C/min to 250 deg.C (keeping for 15 min); sample introduction amount: 1ul.
Mass spectrum conditions: interface temperature 280 ℃, ion source temperature: the ion source (EI) is bombarded by electrons at 230 ℃ to 70eV, and the mass scanning range is 30-600 m/z.
Before the determination, methyl esterification is carried out according to the national standard GB 5009.168-2016:
and operated according to the national food safety standard, namely determination of fatty acids in food.
Preferably, the saponification of fats and methyl esterification of fatty acids is as follows:
adding 8ml of sodium hydroxide methanol solution with the mass concentration of 2% into the fat extract, connecting with a reflux condenser, and refluxing on a water bath at 80 +/-1 ℃ until oil drops disappear. 7ml of a 15% by mass boron trichloride methanol solution (methyl esterification reagent) is added from the upper end of the reflux condenser, and the mixture is continuously refluxed for 2min in a water bath at the temperature of 80 +/-1 ℃. The reflux condenser was flushed with a small amount of water. The heating was stopped, the flask was removed from the water bath and rapidly cooled to room temperature.
Then 10ml-30ml of n-heptane is added accurately, shaking is carried out for 2min, saturated sodium chloride aqueous solution is added, and standing and layering are carried out. Absorbing 5ml of the upper layer n-heptane extraction solution, adding 3g-5g of anhydrous sodium sulfate into a 25ml test tube, shaking for 1min, standing for 5min, and absorbing the upper layer solution into a sample injection bottle to be measured.
33 compounds were identified from rhododendron leaf oil and rhododendron branch oil (see table 3), and the total ion flow diagrams thereof are shown in fig. 1 and fig. 2. The leaf oil contains 22 compounds, the highest of which is methyl linoleate (36.61%), the second is methyl linolenate (23.84%), and the rest compounds with higher content (> 5%) are sequentially arranged according to the content order: methyl 11 cis-octadecenoate (9.57%) > methyl palmitate (8.56%) > methyl tetracosanoate (7.60%). 29 compounds were identified in the tall oil, with the highest content being delta-cnidium (15.15), followed by heptacosane (13.84%), and the others with higher content (> 5%) in the order of their content: gamma-eucalyptol (7.38%) > 4-phenyl-2-butanone (6.63%) > methyl tetracosanoate (6.60%) > methyl linolenate (6.22%) > methyl linoleate (5.81%).
TABLE 3 GC-MS content measurement results
Note: "-" represents no detection
Because the quantitative determination of the fatty acid by GC-MS requires methyl esterification, the contents of the methyl ester compounds in the table are the contents of the corresponding fatty acids.
Example 2 antioxidant Activity assay
DPPH scavenging experiment
0.0197g of DPPH is weighed, absolute ethyl alcohol is used for fixing the volume to 250mL, a solution with the concentration of 0.2mmol/mL is prepared, and the solution is stored at 4 ℃.
2.8045g of rhododendron leaf oil and rhododendron branch oil are respectively weighed and prepared into a series of solutions with the concentrations (mg/mL) of 0.1875, 0.375, 0.75, 1.5, 3, 6, 10 and 12 by using absolute ethyl alcohol as a solvent.
Respectively measuring 2mL of rhododendron leaf oil solution and rhododendron branch oil solution with different concentrations, mixing with 2mL of DPPH solution in a test tube, and reacting for 30min and 60min in a dark place. The reacted reagent was pipetted into a 96-well plate and the absorbance value was read at 517 nm.
2mL of absolute ethanol and 2mL of DPPH reaction solution were used as blank control.
Percent clearance%
And (3) measuring results: the DPPH clearance of the rhododendron leaf oil and the rhododendron branch oil was measured at 30min and 60min, respectively, as shown in FIGS. 10-13.
2. ABTS method for Total Oxidation Capacity
2.8040g of rhododendron leaf oil and rhododendron branch oil are respectively weighed, absolute ethyl alcohol is used as a solvent, and the mixture is prepared into a series of solutions with the concentration (mg/mL) of 200, 150, 120, 96, 48, 24, 12, 6, 3 and 1.5.
The reagent III comprises the following components in percentage by weight: 39, adding distilled water (namely 40 times) to prepare application liquid which is prepared for use. According to the proportion of reagent I: and a second reagent: preparing a working solution from a reagent three application solution (76: 5: 4), wherein the reagent one is a detection buffer solution, the reagent two is an ABTS solution, the reagent three is a peroxide solution, and the reagent four is peroxidase. Storing at room temperature in dark place, and finishing use within 30 min. Reagent four the application liquid is as follows: the first reagent is prepared at a ratio of 1:9 (i.e. 10 times), and is used as it is.
Blank controls were: 10uL of water, 20uL of reagent four application liquid and 170uL of working solution.
The test tube was: 10uL sample +20uL reagent four application solution +170uL working solution.
The reaction was carried out at room temperature for 6min, and the OD value was measured at 439 nm.
Percent clearance%
Standard curve: y (corresponding to the volume of the standard solution, namely the strong and weak oxidation resistance) — 1.1214x (OD value) +1.1262R2 ═ 0.9957;
therefore, the larger the OD value, the smaller the Y value, and the weaker the antioxidant ability.
TABLE 1 ABTS method operation chart of total oxidation capacity
And (3) testing results: the antioxidant capacity of rhododendron leaf oil and rhododendron branch oil in ABTS experiment was measured separately, as shown in FIG. 14 and FIG. 15.
FRAP experiment
2.8040g of rhododendron leaf oil and rhododendron branch oil are respectively weighed and prepared into a series of solutions with the concentration (mg/mL) of 200, 120, 96, 75, 60, 48, 30, 24, 12 and 3 by using absolute ethyl alcohol/petroleum ether (1:1) as a solvent.
Preparing an FRAP working solution according to the following reagent I: and a second reagent: the reagent is prepared in a ratio of 10:1:1, and is prepared in situ.
Control group: 5uL of distilled water +180uL of FRAP working solution.
Measurement group: 5uL of sample solution +180uL of FRAP working solution.
Reacting at 37 deg.C for 3-5min, and measuring OD value at 593 nm.
Percent clearance ═ 1-a sample/a blank x 100%.
TABLE 2 FRAP Experimental operation Table
The experimental results are as follows: the antioxidant capacity of rhododendron leaf oil and rhododendron branch oil in ABTS experiment was measured separately, as shown in FIG. 16 and FIG. 17.
And (3) analyzing the test result of the antioxidant capacity:
in DPPH clearance experiment, the DPPH clearance rate reaches the maximum at the concentration of 6mg/ml when the rhododendron leaf oil and the rhododendron branch oil are both 30 min. Wherein the removal rate of DPPH of the rhododendron leaf oil is 93.9%, and the removal rate of rhododendron branch oil is 90.5%.
In ABTS experiment, the maximum oxidation resistance of rhododendron leaf oil is 0.740, and the corresponding test concentration is 150 mg/ml; the maximum antioxidant capacity of the rhododendron oil is 0.716, corresponding to a test capacity of 200 mg/ml.
In FRAP experiment, the maximum oxidation resistance of rhododendron leaf oil is 0.908, and the corresponding test concentration is 150 mg/ml; the maximum antioxidant capacity of the rhododendron oil is 0.898, corresponding to a test capacity of 200 mg/ml.
Compared with the oxidation resistance of the rhododendron leaf oil and the rhododendron branch oil by three different oxidation resistance measuring methods, the DPPH clearance of the rhododendron leaf oil is higher, the difference between the rhododendron leaf oil and the rhododendron branch oil is not obvious in an ABTS experiment and an FRAP experiment, and the rhododendron branch oil also has good oxidation resistance.
Claims (3)
1. A method for identifying rhododendron anthopogonoide branch oil and leaf oil is characterized in that whether the rhododendron anthopogonoide extract contains the following marker compounds or not is detected: methyl linoleate, methyl linolenate, delta-cnidium lactone, 4-phenyl-2-butanone, cadinene, 3, 9-cadiene, 4(14),7(11) -eucalyptene, eicosane, tetracosane, 2-heptadecanone, hexacosane, 2-nonadecanone, nonacosane and Turkish pimento ketone, and if the compounds are contained at the same time, judging the rhododendron oil; or detecting whether the rhododendron anthopogonoides extract simultaneously contains the following marker compounds: methyl linoleate, methyl linolenate, delta-cnidium lactone, 4-phenyl-2-butanone, cadinene, 3, 9-cadiene, 4(14), (7), (11) -eucalyptene, 2-heptadecanone, 2-nonadecanone and myrtle ketone, and if the compounds are contained at the same time, judging the rhododendron branch oil to be the rhododendron branch oil;
or detecting whether the rhododendron anthopogonoides extract simultaneously contains the following marker compounds: methyl linoleate, methyl linolenate, delta-cnidium lactone, 4-phenyl-2-butanone, methyl dodecanoate, 1(10)6, 8-cadotriene, methyl heptadecanoate and methyl oleate, and if the methyl linoleate, the methyl linolenate, the delta-cnidium lactone, the methyl 4-phenyl-2-butanone, the methyl dodecanoate and the methyl oleate are contained simultaneously, the rhododendron leaf oil is judged.
2. The method of claim 1, wherein the compound is detected by a gas chromatography-mass spectrometry method, and the method comprises the following conditions:
gas chromatography conditions: the sample inlet temperature is 240 ℃, and the column temperature adopts programmed temperature rise: maintaining the initial temperature at 40 deg.C for 5min, and increasing to 250 deg.C at 4 deg.C/min for 15 min; a detector: MSD, detector temperature 230 ℃; carrier gas N2The flow rate is 1.0 mL/min; the split injection split ratio is 10: 1;
mass spectrum conditions: the interface temperature is 280 ℃, the ion source temperature is 230 ℃, the electron bombardment ion source (EI) is 70eV, and the mass scanning range is 30-600 m/z.
3. The method of claim 2, further comprising the following condition:
chromatographic conditions are as follows: column DB-5MS column (30m × 0.25mm × 0.25 um); sample injector: PSS; carrier gas: high purity helium gas; the column flow rate is 1.0 mL/min; sample introduction amount: 1 ul;
mass spectrum conditions: the instrument comprises the following steps: thermo Trace1300/8000Evo GC/MS/MS gas phase tandem mass spectrometer.
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