A method for rapid and simultaneous detection of phenolic and triterpenoid compounds in olive processing by-products

Abstract

The present invention discloses an analytical method for simultaneously and rapidly detecting phenolic and triterpenoid compounds in olive processing byproducts. The rapid detection method of the present invention comprises the following two steps: 1) raw material pretreatment: using olive processing byproducts (leaves, branches, pomace, etc.) as raw materials, naturally air-dried to a moisture content of no more than 5% away from sunlight, crushed to about 0.42 mm, adding an appropriate volume of ethanol-water of a certain concentration as an extraction solvent for simultaneously enriching phenolic and triterpenoids, extracting for a period of time under the assistance of ultrasound, filtering while hot, and passing through a 0.45 μm organic filter membrane after constant volume with an alcohol-water solvent, and the obtained filtrate is used as a subsequent test. 2) Liquid chromatography-mass spectrometry (HPLC-DAD-MS) detection: Under appropriate chromatographic and other detection conditions, it is possible to simultaneously detect a variety of phenolic and triterpenoid compounds in olive processing byproducts. Specifically, Agilent's Poroshell 120, EC-C18 (150mm×3.0mm id, 2.7μm particle size) chromatographic column was used, mobile phase A was acetonitrile and mobile phase B was distilled water containing acidic components, and binary gradient elution was performed. The elution gradient program was as follows: acetonitrile (solvent A) started from 5% at 0.1min, changed from 5% to 40% at 40min, maintained at 40% to 45min, then increased to 70% at 50min, maintained at 70% to 60min; increased to 100% at 65min, maintained at 100% until 68min, and finally returned to 5% at 70min. The detection wavelengths were set to 210, 240, 280 and 350nm, and phenolic and triterpenoid compounds in the sample solution could be successfully detected at a certain column temperature and a certain flow rate. The detection method of the present invention has the characteristics of simplicity, easy operation, high efficiency and wide applicability, and can also be used to simultaneously detect phenolic and triterpenoid compounds in processing by-products of olives of different varieties.

Description

Technical Field

[0001] The invention relates to the field of detecting multiple effective components in natural products, and in particular to a method for simultaneously and rapidly detecting phenolic and triterpenoid compounds in olive processing byproducts. Background Art

[0002] The distribution of phenolic compounds in different olive tree tissues (such as leaves, branches or twigs) and byproducts of olive oil production has always been different, mainly depending on the presence of specific secreted iridoids and glycosylated molecules. Leaves contain a large number of phenolic compounds such as oleuropein and verbenaside, as well as flavonoids such as luteolin and apigenin. Olive pomace has been found to be rich in hydroxytyrosol and verbascoside (Servili et al., 1999). Among olive tissues, seeds and branches have been less studied, and salidroside and nüzhenide have been isolated only in olive seeds (Servili, Baldioli, Selvaggini, Macchioni, & Montedoro, 1999). As for terpenoids, olive leaves and pomace are rich in maslinic acid and oleanolic acid. For example, the content of oleanolic acid in leaves is between 3.0% and 3.5%, followed by maslinic acid and ursolic acid, erythrodiol and uvaol (Guinda, Rada, Delgado, Gutiérrez Adánez and Castellano, 2010). Olive processing waste contains rich active substances such as schizoterpenoids (oleuropein, etc.), polyphenols (hydroxytyrosol, etc.), triterpene acids (maslinic acid, etc. and flavonoids), which can protect it from pathogens and insects, and have pharmacological activities beneficial to human health such as antioxidant, anti-inflammatory, antibacterial, antiviral, anticancer and prevention of cardiovascular diseases, etc., and can be widely used in functional foods and medicine. In general, the traditional use of HPLC can only determine a certain type of substance at a time, and the determination of multiple compounds often requires multiple settings of chromatographic and mass spectrometric conditions and multiple experiments. For example, oleuropein, as a polyphenolic substance of isocyanate ether terpenoids, can be detected by HPLC using a C18 ODS column, methanol-water (0.2% acetic acid) at 230nm to detect the content of oleuropein in olive leaves (Wang Chengzhang et al., 2008, Chemistry and Industry of Forest Products). Some researchers also used HPLC to simultaneously determine polyphenolic compounds such as oleuropein and hydroxytyrosol in olive leaves at 230nm. The specific conditions were: the chromatographic column was ZORBAX Eclipse, XDB-C18 (250×4.6mm, 5μm), the mobile phase was 100% methanol and 0.2% phosphoric acid solution, the flow rate was 0.6mL / min, the time was 30min, and the column temperature was 30℃ (Liu Min, 2019, Nanjing Forestry University). The contents of maslinic acid and oleanolic acid in olive pomace can be successfully determined by HPLC. The specific conditions are as follows: detection wavelength: 210 nm, AT LiCHROM C18 chromatographic column (250 mm × 4.6 mm, 5 μm; mobile phase: methanol-1% acetic acid aqueous solution (v:v = 91:9), flow rate: 1.0 mL min -1, column temperature: 30 ° C, injection volume: 10 μL, manual injection. Before the sample is injected, the solution is filtered with a 0.45 μm microporous filter membrane (Wang Zhu, 2012, Lanzhou University of Technology). The determination of triterpenoids such as ursolic acid, oleanolic acid and erythrodiol in pomace oil can be accurately detected by HPLC (Maria et al., Current Nutrition & Food Science, 2006, 2 (1): 45-49). However, there are few reports on the simultaneous determination of polyphenols and pentacyclic triterpenoids (oleanane type) in olive processing byproducts such as leaves, pomace, branches, etc. by HPLC-DAD-MS. Summary of the invention

[0003] The invention provides an analytical method for simultaneously and rapidly detecting phenolic and triterpenoid compounds in olive processing by-products, which has the characteristics of simplicity, high efficiency, and easy operation.

[0004] The present invention also provides a method, which can be specifically applied to olive processing by-products such as leaves, pomace, branches, core shells, core kernels, etc. The target compounds detected include oleuropein, hydroxytyrosol, tyrosol, caffeic acid, ferulic acid, coumaric acid, luteolin, luteolin glycoside, apigenin, apigenin, quercetin, verbascoside, hawthorn acid, ursolic acid, oleanolic acid, erythrodiol and uvaol, etc.

[0005] The method for simultaneously and rapidly detecting phenolic and triterpenoid compounds in olive processing byproducts of the present invention comprises the following technical schemes: 1) raw material pretreatment: using olive processing byproducts (leaves, branches, pomace, etc.) as raw materials, drying them naturally in the dark and then crushing them, adding an appropriate amount of ethanol-water as extraction solvent, performing ultrasonic assisted extraction for a period of time at room temperature, filtering them while hot, fixing the volume and then passing through a 0.45 μm organic filter membrane, and the obtained filtrate is used for subsequent detection. 2) determination by liquid chromatography-mass spectrometry (HPLC-DAD-MS): using Agilent's Poroshell 120, EC-C18 (150 mm×3.0 mm id, 2.7 μm particle size) chromatographic column as the stationary phase, selecting an appropriate binary organic solvent as the mobile phase at an appropriate column oven temperature, controlling the flow rate and injection volume of the liquid chromatography under appropriate elution program conditions, and detecting the sample solution at multiple appropriate ultraviolet wavelengths. The detection method of the invention can be used to simultaneously detect organic acids, flavonoids, secoiridoid ether terpenes, lignin phenols, pentacyclic triterpenes (oleanane type) and other compounds in by-products of olive processing of different varieties.

[0006] The varieties of the olive processing by-product raw materials (leaves, branches, pomace, etc.) are one or more of Foao, Laixing, Moyola, Pixili, Asi, Peiduoling, Ezhi No. 8 and Chenggu No. 32.

[0007] The air-drying and crushing of the olive processing byproduct raw materials (leaves, branches, pomace, etc.) means that the raw materials are air-dried until the moisture content does not exceed 5%, and the crushed particle size is about 0.42 mm.

[0008] The adding of an appropriate amount of ethanol-water as a solvent refers to an ethanol-water volume ratio in the range of 70% to 90%; the ultrasonic-assisted extraction for a period of time refers to an ultrasonic power of 50 to 300 W and an extraction time of 5 to 30 minutes; the dried raw material powder and the extraction solvent ratio (g / mL) are filtered while hot, constant in volume, and then passed through a 0.45 μm organic filter membrane, and the obtained filtrate is prepared for subsequent detection.

[0009] The power parameters of the ultrasonic-assisted rapid extraction are 100-2000W, the extraction time is 0.5-100min, and the solid-liquid ratio (g / mL) is 1:5-1:50.

[0010] The appropriate column oven temperature is 25-35° C., and the mobile phase of the binary organic solvent is acetonitrile (A) and distilled water containing an acidic component (pH=3.2) (B), and the acidic component is one of formic acid, acetic acid, phosphoric acid and hydrochloric acid.

[0011] The appropriate elution program conditions refer to the following conditions: 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; increases to 100% at 65 min, remains at 100% until 68 min, and finally returns to 5% at 70 min.

[0012] The control of the flow rate and injection volume of the liquid chromatography refers to a flow rate of 0.4 to 1.0 mL min -1 , the injection volume is 5~20μL.

[0013] The multiple suitable ultraviolet wavelengths mentioned are 210, 240, 280 and 350 nm.

[0014] Beneficial Effects

[0015] The present invention provides an analytical method for simultaneously and rapidly detecting phenolic and triterpenoid compounds in olive processing byproducts, which is simple to operate, safe and efficient, and can simultaneously determine multiple different types of active compounds such as organic acids, flavonoids, secoiridoids, lignin phenols, pentacyclic triterpenes (oleanane type) and other compounds. The present invention can be used for multiple determinations of different types of compounds, which can significantly reduce the consumption of experimental reagents, reduce experimental time and experimental costs, and improve work efficiency.

[0016] Description of the drawings (hereinafter referred to as Figure 1-6 All are Laixing varieties)

[0017] Figure 1 HPLC spectrum of phenolic compounds in olive leaves.

[0018] Figure 2 This is the HPLC spectrum of phenolic compounds in olive branches.

[0019] Figure 3 HPLC spectrum of phenolic compounds in olive pomace.

[0020] Figure 4 This is the HPLC spectrum of phenolic compounds in olive kernel and shell.

[0021] Figure 5 This is the HPLC spectrum of phenolic compounds in olive kernel.

[0022] Figure 6 HPLC spectrum of triterpenoids in olive leaves (A, Maslinic acid; B, Oleanolic acid; C, Ursolic acid; D, Erythrodiol.

[0023] Remark: Figure 1-Figure 5, the compounds corresponding to Compound Numbers 1 to 43 are as follows: 1, hydroxytyrosol; 2, taxifolin glucoside isomer; 3, esculetin; 4, eriodictyol-7-O-glucoside isomer; 5, verbascoside; 6, luteolin-7-O-glucoside; 7, taxifolin isomer; 8, oleuropeindiglucoside(+comselogoside isobar); 9, 1-acetoxypinoresinol glucoside; 10, eriodictyol isomer; 11, demetyloleuropein; 12, rutin; 13, nüzhenide; 14, cafselogoside; 15, oleuropein; 16, comselogoside; 17, oleuropein aglycone; 18, ligstroside; 19, caffeic acid; 20, luteolin-3,7-diglucoaside; 21, luteolinderivative; 22, apigenin glucuronide; 23, luteolin-4-O’-glucoside; 24, hydroxytyrosol glucoside; 25, tyrosol; 26, β-OH acteoside 1; 27, β-OH acteoside; 28, luteolin; 29, tyrosol derivatives; 30, oleoside11-methyl ester; 31, oleoside 11-methyl ester isomer; 32, nüzhenide derivative; 33, bis(oleoside11-methyl ester)glucoside; 34, salidroside oleoside; 35, nüzhenide isomer; 36, nüzhenide 11-methyloleoside isomers 1-3; 37, nüzhenide 11-methyl oleoside; 38, nüzhenide di-(11-methyloleoside)isomers 1-2; 39, ligstroside oleoside;40, lariciresinol-sesquilignan+hydroxytyrosol acetate; 41, cinnamic derivative; 42, pinoresinol; 43, secoiridoid. ;

[0024] Example 1

[0025] ① Raw material pretreatment: Preparation of olive leaf extract

[0026] 1.0 g of Frantoio olive leaves picked in November and dried naturally away from sunlight with a water content of 4.5% were crushed to 0.4 mm and added to 30 mL of 90% ethanol-water in a beaker. The mixture was extracted for 30 min at room temperature with an ultrasonic power of 300 W. The filtrate was filtered while hot to obtain a filtrate that was fixed to 100 mL and filtered through a 0.45 μm organic filter membrane before entering HPLC.

[0027] ② Simultaneous detection of phenolic and triterpenoid compounds in olive leaf extract by HPLC-DAD-MS

[0028] The olive leaf extract prepared above was chromatographically analyzed using a Poroshell 120, EC-C18 (150 mm × 3.0 mm id, 2.7 μm particle size) column from Agilent Technologies at 26°C. The mobile phase was acetonitrile (A) and distilled water containing formic acid (pH = 3.2) (B), with a flow rate of 0.4 mL min -1 , the injection volume was 5μL. The detection wavelengths were 210, 240, 280 and 350nm, respectively. A started from 5% at 0.1min, changed from 5% to 40% at 40min, remained at 40% to 45min, then increased to 70% at 50min, remained at 70% to 60min; increased to 100% at 65min, remained at 100% until 68min, and finally returned to 5% at 70min. Phenolic compounds and triterpenoid compounds can be successfully detected, and the specific test results are shown in the following table.

[0029]

[0030] Example 2

[0031] ① Preparation of olive pomace extract:

[0032] 2.0 g of olive pomace with a water content of 4.8% was collected from the olive oil processing plant Leccino, crushed to 0.42 mm, added with 50 mL of 85% ethanol-water in a beaker, extracted for 20 min at room temperature with an ultrasonic power of 500 W, filtered while hot to obtain the filtrate, fixed to 250 mL, and passed through a 0.45 μm organic filter membrane before entering HPLC.

[0033] ②HPLC-DAD-MS simultaneous detection of phenolic and triterpenoid compounds in olive pomace extract:

[0034] A Poroshell 120, EC-C18 (150 mm × 3.0 mm id, 2.7 μm particle size) column from Agilent Technologies was used at 28°C. The mobile phase was acetonitrile (A) and distilled water containing formic acid (pH = 3.2) (B), with a flow rate of 0.6 mL min -1 , the injection volume was 10μL. The detection wavelengths were 210, 240, 280 and 350nm, respectively. A started from 5% at 0.1min, changed from 5% to 40% at 40min, remained at 40% to 45min, then increased to 70% at 50min, remained at 70% to 60min; increased to 100% at 65min, remained at 100% until 68min, and finally returned to 5% at 70min. Phenolic compounds and triterpenoid compounds can be successfully detected, and the specific test results are shown in the following table.

[0035]

[0036] Example 3

[0037] ① Preparation of olive branch extract:

[0038] 1.5 g of Moraiolo olive branches picked in November and naturally dried away from sunlight with a water content of 4.5% were crushed to 0.4 mm and added to 45 mL of 80% ethanol-water in a beaker. The extraction was carried out at room temperature with an ultrasonic power of 600 W for 20 min. The filtrate was filtered while hot to obtain a filtrate, which was fixed to 250 mL and passed through a 0.45 μm organic filter membrane before HPLC.

[0039] ② HPLC-DAD-MS simultaneous detection of phenolic and triterpenoid compounds in olive branch extract:

[0040] The olive branch extract prepared above was chromatographically separated using a Poroshell 120, EC-C18 (150 mm × 3.0 mm id, 2.7 μm particle size) column from Agilent Technologies at 30°C. The mobile phase was acetonitrile (A) and distilled water containing formic acid (pH = 3.2) (B), with a flow rate of 1.0 mL min -1 , the injection volume was 20μL. The detection wavelengths were 210, 240, 280 and 350nm, respectively. A started from 5% at 0.1min, changed from 5% to 40% at 40min, remained at 40% to 45min, then increased to 70% at 50min, remained at 70% to 60min; increased to 100% at 65min, remained at 100% until 68min, and finally returned to 5% at 70min. Phenolic compounds and triterpenoid compounds can be successfully detected, and the specific test results are shown in the following table.

[0041]

Claims

1. An analytical method for simultaneously and rapidly detecting phenolic and triterpenoid compounds in olive processing by-products, characterized in that: 1) Raw material pretreatment: The byproducts of olive processing are used as raw materials, dried naturally in the dark and then crushed, ethanol-water is added as extraction solvent, ultrasonic-assisted extraction is performed at room temperature, and the mixture is filtered while hot and fixed to volume before passing through a 0.45 μm organic filter membrane. The filtrate obtained is used for subsequent detection; 2) Determination is performed by liquid chromatography-mass spectrometry; Agilent's Poroshell 120, EC-C18 chromatographic column is used as the stationary phase, and its specifications are: 150 mm × 3.0 mm id, 2.7 μm particle size; at a column temperature of 25-35° C., a binary organic solvent consisting of a mobile phase A of acetonitrile and a mobile phase B of a distilled water solution containing formic acid and a mobile phase B solution with a pH of 3.2 is selected as the mobile phase; the liquid phase elution program conditions of the mobile phase of the binary organic solvent are as follows: the mobile phase A starts from 5% at 0.1 min, changes from 5% to 40% at 40 min, is maintained at 40% to 45 min, then increases to 70% at 50 min, is maintained at 70% to 60 min; increases to 100% at 65 min, is maintained at 100% until 68 min, and finally returns to 5% at 70 min; the flow rate and injection volume of the liquid chromatography are controlled, and the standby filtrate is detected at ultraviolet absorption wavelengths of 210, 240, 280 and 350 nm; the detection method of the invention can be used to simultaneously detect the content of phenolic and triterpenoid compounds in olive processing by-products of different varieties.

2. The method for simultaneously and rapidly detecting phenolic and triterpenoid compounds in olive processing byproducts according to claim 1, characterized in that: The varieties of the olive processing by-product raw materials are one or more of Foao, Laixing, Moyola, Picili, Asi, Peiduoling, Ezhi No. 8 and Chenggu No.

32.

3. The method for simultaneously and rapidly detecting phenolic and triterpenoid compounds in olive processing byproducts according to claim 1, characterized in that: The air-drying and crushing of the olive processing byproduct raw material refers to air-drying until the moisture content of the raw material does not exceed 5%, and the crushed particle size is about 0.42mm.

4. The method for simultaneously and rapidly detecting phenolic and triterpenoid compounds in olive processing byproducts according to claim 1, characterized in that: The adding of an appropriate amount of ethanol-water as a solvent means that the volume ratio of ethanol-water is in the range of 70% to 90%; the power parameter of ultrasonic-assisted extraction is 100 to 2000W, the extraction time is 0.5 to 100 minutes, the ratio of raw material mass to solvent liquid volume is 1:5 to 1:50, the mass unit is g, and the liquid volume unit is mL.

5. The method for simultaneously and rapidly detecting phenolic and triterpenoid compounds in olive processing byproducts according to claim 1, characterized in that: The control of the flow rate and injection volume of the liquid chromatography refers to a flow rate of 0.4 to 1.0 mL / min and an injection volume of 5 to 20 microliters.

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