CN113884590A - Analysis method capable of simultaneously and rapidly detecting phenols, triterpenes and other compounds in olive processing by-products - Google Patents

Abstract

Translated fromChinese

本发明公开了一种可同时快速检测油橄榄加工副产物中酚类和三萜类等多种化合物的分析方法。本发明的快速检测方法包含以下两个步骤：1)原料预处理：以油橄榄加工副产物（叶、枝、果渣等）为原料，自然风干后粉碎，乙醇‑水作为提取溶剂，在超声波辅助下提取一段时间，经抽滤、乙醇‑水定容后过滤膜，获滤液后待检测。2)HPLC‑DAD‑MS检测：采用反相色谱柱进行二元梯度洗脱，流动相为乙腈(A)和含少量酸的蒸馏水(B)，在一定柱温、流速和多波长设定下，进样适量体积样品溶液后进行检测。本发明方法具有简易操作、高效和适用性广等特点，也可用于同时检测不同品种油橄榄加工副产物中有机酸、黄酮、裂环烯醚萜类、木素酚类、五环三萜类（齐墩果烷型）等化合物。The invention discloses an analysis method which can simultaneously and rapidly detect various compounds such as phenols and triterpenes in by-products of olive processing. The rapid detection method of the present invention comprises the following two steps: 1) raw material pretreatment: using olive processing by-products (leaf, branch, pomace, etc.) as raw materials, pulverizing after natural air-drying, ethanol-water as an extraction solvent, assisted by ultrasonic waves After extracting for a period of time, the membrane is filtered after suction filtration and ethanol-water constant volume, and the filtrate is obtained for testing. 2) HPLC‑DAD‑MS detection: binary gradient elution was performed on a reversed-phase chromatographic column, and the mobile phase was acetonitrile (A) and distilled water (B) containing a small amount of acid, under certain column temperature, flow rate and multi-wavelength settings , and test after injecting an appropriate volume of sample solution. The method of the invention has the characteristics of simple operation, high efficiency and wide applicability, and can also be used for simultaneous detection of organic acids, flavonoids, split iridoids, lignophenols, pentacyclic triterpenes ( oleanane type) and other compounds.

Description

Analysis method capable of simultaneously and rapidly detecting phenols, triterpenes and other compounds in olive processing by-products

Technical Field

The invention relates to the field of detection of various active ingredients in natural products, in particular to a method for simultaneously and rapidly detecting various compounds such as phenols, triterpenes and the like in olive processing byproducts.

Background

The distribution of phenolic compounds in different olive tree tissues (such as leaves, branches or twigs) and olive oil production by-products is always different, mainly depending on the specific secretory ringThe presence of alkenyl ether terpenes and glycosylated molecules. The leaves contain a large amount of phenolic compounds, such as oleuropein and verbascoside, and flavonoids such as luteolin and apigenin. It has been found that olive pomace is rich in hydroxytyrosol and verbascoside (Servili et al, 1999). In olive tissue, seeds and shoots are less studied, salidroside and nzhenide are only isolated in olive seeds (Servili, baldiili, selvagini, Macchioni and Montedoro, 1999). As for terpenoids, olive leaves and pomace are rich in maslinic acid and oleanolic acid. For example, the oleanolic acid content in leaves is between 3.0% and 3.5%, followed by maslinic and ursolic acid, kaegol and uvaol (guida, Rada, Delgado, Guti erez Ad nez and Castellano, 2010). The olive processing waste contains rich active substances such as seco-iridoid (oleuropein, etc.), polyphenol (hydroxytyrosol, etc.), triterpenic acid (crataegolic acid, etc. and flavonoid), can prevent the olive processing waste from being invaded by germs and insects, has pharmacological activities beneficial to human health, such as oxidation resistance, anti-inflammation, antibiosis, antivirus, anticancer, cardiovascular disease prevention, etc., and can be widely applied to the fields of functional foods, medicines, etc. In general, a certain substance can be measured by using HPLC in a single time, and the measurement of a plurality of compounds usually requires setting chromatographic and mass spectrum conditions for a plurality of times and executing a plurality of experiments. For example, oleuropein as a iridoid polyphenol substance in the form of a cyclic enol ether can be detected by HPLC using a C18 ODS column, methanol-water (0.2% acetic acid), and the oleuropein content in the olive leaf species can be detected at 230 nm (Wangchang et al, 2008, Lin chemical and Industrial). Researchers have also measured olive leaf polyphenol compounds such as oleuropein and hydroxytyrosol simultaneously at 230 nm by HPLC, with the specific conditions: the column was ZORBAX Eclipse, XDB-C18 (250X 4.6 mm, 5 μm), the mobile phase was 100% methanol and 0.2% phosphoric acid solution, the flow rate was 0.6 m L/min, the time was 30 min, and the column temperature was 30 ℃, (Liumin, 2019, university of Nanjing forestry). The contents of maslinic acid and oleanolic acid in the olive pomace can be successfully determined by HPLC, and the specific conditions are as follows: 210 nm AT LiCHROM C18 chromatographic column (250 mm × 4.6 mm, 5 μm; mobile phase is methanol-1% acetic acid water solution(v: v =91: 9), flow rate: 1.0 mL/min^-1Column temperature: 30 ℃, sample introduction: 10 μ L, sample injection by hand. The solution before the sample to be tested was filtered through a 0.45 μm microporous membrane (Wang, 2012, university of Lanzhou Rich). The determination of triterpene substances such as ursolic acid, oleanolic acid and homodiol in the fruit residual oil can be accurately detected by HPLC (Maria et al.,. Current Nutrition)&Food Science, 2006, 2(1): 45-49). However, it is only rarely reported that by HPLC-DAD-MS is used to simultaneously produce polyphenols, organic acids, iridoids, pentacyclic triterpenes (oleanane type) in olive processing by-products such as leaves, pomace, branches, etc.

Disclosure of Invention

The invention provides an analysis method capable of simultaneously and rapidly detecting phenols, triterpenes and other various compounds in olive processing byproducts, and the analysis method has the characteristics of simplicity, convenience, high efficiency, easiness in operation and the like.

The invention also provides a method which can be specifically applied to olive processing byproducts such as leaves, pomace, branches, nutshells and the like, and the detected target compounds comprise oleuropein, hydroxytyrosol, tyrosol, caffeic acid, ferulic acid, coumaric acid, luteolin, apigenin, quercetin, verbascoside, maslinic acid, ursolic acid, oleanolic acid, homogen diol, Uvaol (Uvaol) and the like.

The method for simultaneously and rapidly detecting phenols, triterpenes and other various compounds in the olive processing by-products comprises the following technical scheme: 1) pretreatment of raw materials: taking olive processing byproducts (leaves, branches, pomace and the like) as raw materials, naturally drying in the sun and then crushing, adding a proper amount of ethanol-water as an extraction solvent, carrying out ultrasonic-assisted extraction for a period of time at room temperature, carrying out suction filtration while hot, fixing the volume and then passing through a 0.45-micrometer organic filter membrane to obtain filtrate for subsequent detection. 2) Determination by liquid chromatography-mass spectrometry combined technique (HPLC-DAD-MS): the method is characterized in that Poroshell 120, EC-C18 (150 mm multiplied by 3.0 mm id, 2.7 mu m particle size) chromatographic columns of Agilent company are used as stationary phases, proper binary organic solvents are selected as mobile phases at proper column incubator temperature, the flow rate and the sample injection quantity of liquid chromatography are controlled under proper elution program conditions, and sample solutions are detected under multiple proper ultraviolet wavelengths. The detection method can be used for simultaneously detecting the compounds such as organic acid, flavone, secoiridoid, lignin phenol, pentacyclic triterpenes (oleanane type) and the like in the processing byproducts of different varieties of olives.

The variety of the olive processing by-product raw materials (leaves, branches, pomace and the like) is one or more of Foao, Luixing, Mozula, Pieli, Asi, Bigduoling, Hubei plant No. 8 and City fixed No. 32.

The air drying and crushing of the olive processing by-product raw materials (leaves, branches, pomace and the like) means that the raw materials are air dried until the water content of the raw materials is not more than 5 percent, and the crushed granularity is about 0.42 mm.

The addition of a proper amount of ethanol-water as a solvent means that the volume ratio of ethanol-water is 70-90%; the ultrasonic-assisted extraction is carried out for a period of time, wherein the ultrasonic power is 50-300W, and the extraction time is 5-30 min; the dried raw material powder and the extraction solvent are subjected to suction filtration while hot in proportion (g/mL), and filtered through a 0.45-micron organic filter membrane after constant volume, and the obtained filtrate is prepared for subsequent detection.

The power parameter of the ultrasonic-assisted rapid extraction is 100-2000W, the extraction time is 0.5-100 min, and the material-liquid ratio (g/mL) is 1: 5-1: 50.

The proper temperature of the column oven is 25-35 ℃, the mobile phase of the binary organic solvent is acetonitrile (A) and distilled water (pH = 3.2) (B) containing acid components, and the acid components are one of formic acid, acetic acid, phosphoric acid and hydrochloric acid.

Under the condition of proper elution procedure, the following conditions are referred to: solvent A starts from 5% at 0.1 min, changes from 5% to 40% at 40 min, remains at 40% to 45 min, then increases to 70% at 50 min, remains at 70% to 60 min; increasing to 100% at 65 min, maintaining at 100% until 68 min, and finally returning to 5% at 70 min.

The flow rate and the sample injection quantity of the liquid chromatogram are controlled, and the flow rate is 0.4-1.0 mL/min⁻¹The sampling amount is 5-20𝜇L。

The plurality of suitable ultraviolet wavelengths are referred to as 210, 240, 280 and 350 nm.

Advantageous effects

The invention provides an analysis method capable of simultaneously and rapidly detecting phenols, triterpenes and other compounds in olive processing byproducts, which is simple and convenient to operate, safe and efficient, and can simultaneously detect various active compounds of different classes such as organic acids, flavones, seco-iridoids, lignenols, pentacyclic triterpenes (oleanane types) and other compounds in a single time. The invention can measure different types of compounds for many times, can obviously reduce the consumption of experimental reagents, reduce the experimental time and the experimental cost and improve the working efficiency.

Description of the drawings (the following figures 1-6 are all of the species Lexing)

FIG. 1 is an HPLC chromatogram of phenolic compounds in olive leaves.

FIG. 2 is an HPLC chromatogram of phenolic compounds in olive shoots.

FIG. 3 is an HPLC chromatogram of phenolic compounds in olive pomace.

FIG. 4 is an HPLC chromatogram of phenolic compounds in the olive nucleocapsid.

FIG. 5 is an HPLC chromatogram of phenolic compounds in the olive kernel.

FIG. 6 shows HPLC chromatogram of triterpenoids in olive leaves (A, Maslinic acid; B, Oleanolic acid; C, Ursolic acid; D, Erythrodiol).

In FIGS. 1 to 5, the compounds with thenumbers 1 to 43 are as follows: 1, hydroxytocopherol, 2, tetrahydrothiolin glucoside isocyanate, 3, esculetin, 4, eridol-7-O-glucoside isocyanate, 5, verascoside, 6, luteolin-7-O-glucoside, 7, texilin isocyanate, 8, oleuropein diglucoside (+ cornoside), 9, 1-acetoxythionol glucoside, 10, eridol isocyanate, 11, demetolythiolene, 12, rutin, 13, n ü zhenide, 14, califluoroglucoside, 15, oleuropein, 16, seloside, 17, oleuropein lysoglucoside, 18, lithylenesulfide, 19, 20, 23, 25, 23, 25, 3, 25, one, luteolin, 29 tyrosol derivatives, 30, oleoside11-methyl ester, 31, oleoside11-methyl ester, 32, n-zhenide derivative, 33, bis (oleoside11-methyl ester) glucoside, 34, saliside oleoside, 35, n-zhenide isomer, 36, n-zhenide 11-methyl ester inductors 1-3, 37, n-zhenide 11-methyl ester, 38, n-zhenide di- (11-methyl ester) inductors 1-2, 39, lignososide, 40, laricoside-sessile-ketose + dihydrohexeoside, 41, 42, 23, r.

Example 1

Pretreating raw materials: preparation of olive leaf extract

Collected in 11 months and naturally dried by sunlight-resistant Foao (A)Frantoio) 1.0 g of olive leaves with the water content of 4.5 percent, crushing to 0.4 mm, adding 30 mL of ethanol-water with the volume fraction of 90 percent into a beaker, extracting for 30 min at room temperature under the ultrasonic power of 300W, carrying out suction filtration while the solution is hot to obtain filtrate, fixing the volume to 100 mL, and filtering through an organic filter membrane with the diameter of 0.45 mu m before HPLC. ② HPLC-DAD-MS detects phenols and triterpenes compounds in olive leaf extract simultaneously

The olive leaf extract prepared above was subjected to a column chromatography of Poroshell 120, EC-C18 (150 mm. times.3.0 mm id, 2.7 μm particle diameter) by Agilent Technologies at 26 ℃. The mobile phase was acetonitrile (a) and distilled water containing formic acid (pH = 3.2) (B), and the flow rate was 0.4 mL · min⁻¹The sampling amount is 5𝜇And L. The detection wavelengths were 210, 240, 280 and 350 nm, respectively. A starts at 5% at 0.1 min, changes from 5% to 40% at 40 min, remains at 40% to 45 min, then increases to 70% at 50 min, remains at 70% to 60 min, increases to 100% at 65 min, remains at 100% up to 68 min, and finally returns to 5% at 70 min. Phenolic compounds and triterpenoids can be successfully detected, and specific detection results are shown in the following table:

example 2

Preparing an olive pomace extracting solution:

collected from olive oil processing plant laixing (Leccino) 2.0 g of olive pomace with the water content of 4.8 percent, crushing to 0.42 mm, adding 50 mL of ethanol-water with the volume fraction of 85 percent into a beaker, extracting for 20 min at room temperature under the ultrasonic power of 500W, carrying out suction filtration while hot to obtain a filtrate, fixing the volume to 250 mL, and filtering through a 0.45 mu m organic filter membrane before HPLC. ② HPLC-DAD-MS detects phenols and triterpenes compounds in the olive pomace extract at the same time:

a Poroshell 120, EC-C18 (150 mm. times.3.0 mm id, 2.7 μm particle size) column from Agilent Technologies was used at 28 ℃. The mobile phase was acetonitrile (a) and distilled water containing formic acid (pH = 3.2) (B), and the flow rate was 0.6 mL · min⁻¹The sample size is 10𝜇And L. The detection wavelengths were 210, 240, 280 and 350 nm, respectively. A starts at 5% at 0.1 min, changes from 5% to 40% at 40 min, remains at 40% to 45 min, then increases to 70% at 50 min, remains at 70% to 60 min, increases to 100% at 65 min, remains at 100% up to 68 min, and finally returns to 5% at 70 min. Phenolic compounds and triterpenoids can be successfully detected, and specific detection results are shown in the following table:

example 3

Preparing an olive branch extracting solution:

mojolla picked in 11 months and naturally dried in the sun (Moraiolo) 1.5 g of olive branches with water content of 4.5%, pulverizing to 0.4 mm, adding 45 mL of 80% ethanol-water, and performing ultrasonic treatment at room temperatureThe yield is 600W, extraction is carried out for 20 min, suction filtration is carried out while the solution is hot, filtrate is obtained, the volume is determined to be 250 mL, and the solution is filtered through a 0.45 mu m organic filter membrane before HPLC. ② HPLC-DAD-MS detects phenols and triterpenes compounds in the olive branch extract at the same time:

the olive shoot extract prepared above was subjected to a Poroshell 120, EC-C18 (150 mm. times.3.0 mm id, 2.7 μm particle size) column chromatography by Agilent Technologies, Inc., at a temperature of 30 ℃. The mobile phase was acetonitrile (a) and distilled water containing formic acid (pH = 3.2) (B), and the flow rate was 1.0 mL · min⁻¹The sample size is 20𝜇And L. The detection wavelengths were 210, 240, 280 and 350 nm, respectively. A starts at 5% at 0.1 min, changes from 5% to 40% at 40 min, remains at 40% to 45 min, then increases to 70% at 50 min, remains at 70% to 60 min, increases to 100% at 65 min, remains at 100% up to 68 min, and finally returns to 5% at 70 min. Phenolic compounds and triterpenoids can be successfully detected, and specific detection results are shown in the following table:

Claims (9)

1. an analysis method capable of simultaneously and rapidly detecting phenols, triterpenes and other various compounds in olive processing byproducts is characterized by comprising the following steps of 1) raw material pretreatment: taking olive processing byproducts (leaves, branches, pomace and the like) as raw materials, naturally drying in the sun and then crushing, adding a proper amount of ethanol-water as an extraction solvent, carrying out ultrasonic-assisted extraction at room temperature for a period of time, carrying out suction filtration while hot, fixing the volume and then passing through a 0.45-micrometer organic filter membrane to obtain filtrate for subsequent detection; 2) determination by liquid chromatography-mass spectrometry combined technique (HPLC-DAD-MS): the detection method can be used for simultaneously detecting compounds such as organic acid, flavone, secoiridoid, lignin phenol, pentacyclic triterpene (oleanane type) and the like in processing byproducts of different varieties of olive oil by using poroschel 120 and EC-C18 (150 mm multiplied by 3.0 mm id, 2.7 mu m particle size) chromatographic columns of Agilent company as stationary phases, selecting proper binary organic solvents as mobile phases at proper column incubator temperature, controlling the flow rate and sample injection amount of liquid chromatography under proper elution program conditions, and detecting the standby filtrate under a plurality of proper ultraviolet wavelengths.

2. The method according to claim 1, wherein the olive processing by-product raw material (leaves, branches, pomace, etc.) is one or more of Foo, Ricin, Mozura, Pielli, Asi, Bigdalin, Omei No. 8 and City fixed No. 32.

3. The method as claimed in claim 1, wherein the olive processing by-product raw material (leaves, branches, pomace, etc.) is air-dried and pulverized until the water content of the raw material is not more than 5%, and the pulverized particle size is about 0.42 mm.

4. The method according to claim 1, wherein the adding of a proper amount of ethanol-water as a solvent means that the volume ratio of ethanol-water is 70% -90%; the ultrasonic-assisted extraction is carried out for a period of time, wherein the ultrasonic power is 50-300W, and the extraction time is 5-30 min; the dried raw material powder and the extraction solvent are subjected to suction filtration while hot in proportion (g/mL), and filtered through a 0.45-micron organic filter membrane after constant volume, and the obtained filtrate is prepared for subsequent detection.

5. The method according to claim 1, wherein the power parameter of the ultrasonic-assisted rapid extraction is 100-2000W, the extraction time is 0.5-100 min, and the material-liquid ratio (g/mL) is 1: 5-1: 50.

6. The method as claimed in claim 1, wherein the suitable column oven temperature is 25-35 ℃, and the mobile phase of the binary organic solvent is acetonitrile (A) and acid-containing distilled water (pH = 3.2) (B); and the acid is one of formic acid, acetic acid, phosphoric acid and hydrochloric acid.

7. The method according to claim 1, wherein under appropriate elution program conditions, the following conditions are defined: solvent A starts from 5% at 0.1 min, changes from 5% to 40% at 40 min, remains at 40% to 45 min, then increases to 70% at 50 min, remains at 70% to 60 min; increasing to 100% at 65 min, maintaining at 100% until 68 min, and finally returning to 5% at 70 min.

8. The method according to claim 1, wherein the controlling of the flow rate and the sample amount of the liquid chromatography is performed at a flow rate of 0.4-1.0 mL-min⁻¹The sampling amount is 5-20𝜇L。

9. The method of claim 1, wherein the plurality of suitable ultraviolet wavelengths are 210, 240, 280, and 350 nm.

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