Extraction process of willow bark essential oil, antioxidant evaluation method and application of willow bark essential oil

Abstract

The invention relates to the technical field of willow bark essential oil, in particular to an extraction process, an antioxidant evaluation method and application of willow bark essential oil, and the extraction process comprises the following steps: drying willow bark at low temperature, crushing, and extracting by adopting a supercritical carbon dioxide extraction method under the extraction conditions that the front temperature is 30-33 DEG C, the front pressure is 33-35 MPa, the separation pressure is 10-12 MPa, and the extraction time is 45-48 minutes; the medium temperature is 33-35 DEG C, the medium pressure is 35-38 MPa, the separation pressure is 8-10 MPa, and the extraction time is 75-78 minutes; the post-temperature is 33.5-35 DEG C, the post-pressure is 40-42 MPa, the separation pressure is 6-8 MPa, and the post-stage extraction lasts for 95-100 minutes; according to the method, efficient extraction and activity retention of the willow bark essential oil are realized through an innovative low-temperature drying-supercritical carbon dioxide extraction process, and an anti-oxidation action mechanism of the essential oil is comprehensively indicated by combining a multi-dimensional anti-oxidation activity evaluation system. The method not only solves the problems of component loss and activity reduction in a traditional extraction method, but also provides reliable theoretical support for application of essential oil in the fields of cosmetics, health care products, food and the like through simulated digestion experiments and in-vivo activity verification.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of willow bark essential oil, in particular to a willow bark essential oil extraction process, an antioxidant evaluation method and application thereof. BACKGROUND

[0002] Salix is Salicaceae, and is the general term of Salix and Chosenia plants. As one of the main fast-growing tree species in the north temperate zone, Salix is widely distributed in the temperate and cold regions of the northern hemisphere. According to the records in Flora of China, Salix is divided into 37 groups, with 257 wild species, 122 varieties and 33 types, of which 2 / 3 are shrub willows and 1 / 3 are tree willows, accounting for about 50% of the world's willow species. Among them, 179 species are endemic to China. Salix, with the characteristics of strong adaptability, rapid growth and rich types, not only plays an important role in ecological fields such as water control, sand fixation and alkali improvement, and is an important tree species for greening deserts, wastelands, flooded lands and saline-alkali lands, but also is an important component of timber forest and bioenergy forest, which has significant ecological and economic value in ecological protection, industrial raw materials, biological remediation, bioenergy and landscaping.

[0003] From the perspective of medicinal value, willow bark is widely used in Chinese and foreign medicine. The classics such as Shennong's Herbal Classic and Compendium of Materia Medica record that the roots, bark, branches and leaves of willow have the effects of expelling phlegm, clearing heat and resolving toxins, diuresis and wind prevention. External application can treat toothache, and decoction can treat internal bleeding, jaundice and leukorrhoea. Modern research shows that willow bark contains active ingredients such as salicylic acid, flavonoids and terpenes, and has antibacterial, anti-inflammatory and antioxidant effects. Its essential oil can be used as a natural antioxidant in the fields of cosmetics, health products and food. However, the existing technology has obvious deficiencies in the extraction and antioxidant activity evaluation of willow bark essential oil. Traditional extraction methods (such as ethanol extraction and high-temperature drying) are prone to loss of volatile components and degradation of active substances, affecting the yield of essential oil and antioxidant effect. Existing antioxidant evaluation is mostly limited to single index determination in vitro, lacking stability research simulating human digestive environment and in vivo activity verification, and it is difficult to fully reflect the actual application value of essential oil. SUMMARY

[0004] The present application relates to a willow bark essential oil extraction process, an antioxidant evaluation method and application thereof.

[0005] The present application provides the following technical solutions:

[0006] The invention discloses an extraction process for willow bark essential oil, comprising the following steps: drying the willow bark at a low temperature of 45-48°C, crushing the bark into 40-60 meshes, and extracting the oil by supercritical carbon dioxide extraction, wherein the extraction conditions are as follows: front temperature of 30-33°C, front pressure of 33-35 MPa, separation pressure of 10-12 MPa, and extraction for 45-48 minutes; middle temperature of 33-35°C, middle pressure of 35-38 MPa, separation pressure of 8-10 MPa, and extraction for 75-78 minutes; and rear temperature of 33.5-35°C, rear pressure of 40-42 MPa, separation pressure of 6-8 MPa, and rear extraction for 95-100 minutes.

[0007] As a further technical solution, the willow bark includes shrub willow and tree willow.

[0008] As a further technical solution, the tree willows include weeping willows, matsudanax, and 1011 varieties, and the shrub willows include dustpan willows, two willows, and 2345 varieties. Impurities on the bark surface need to be removed before drying.

[0009] As a further technical solution, a method for evaluating the antioxidant activity of the willow bark essential oil obtained by the process comprises:

[0010] In vitro evaluation included simulated gastric and intestinal digestion to determine DPPH free radical scavenging and ABTS free radical scavenging;

[0011] In vivo evaluation: An animal model of oxidative stress was established, and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) content in the liver tissue of the animal model were detected.

[0012] As a further technical solution, the DPPH free radical scavenging comprises:

[0013] The essential oil was prepared into a 0.1-10 mg / mL ethanol solution, mixed with a 0.06-0.6 mmol / LDPPH ethanol solution, reacted in the dark for 30-40 minutes, and the absorbance was measured at 517 nm.

[0014] As a further technical solution, the ABTS free radical scavenging comprises:

[0015] 7 mM A ABTS solution was mixed with 2.45 mM potassium persulfate in the dark for 12 h, diluted and reacted with the essential oil solution for 6 min, and the absorbance was measured at 734 nm.

[0016] As a further technical solution, the in vivo oxidative stress animal model is established by intraperitoneal injection of D-galactose, and antioxidant indicators are detected after oral administration of willow bark essential oil.

[0017] The obtained willow bark essential oil is used in the preparation of antioxidant products, which include cosmetics, health products and foods.

[0018] As a further technical solution, the cosmetic product comprises a facial mask, facial cleanser, soap, shampoo.

[0019] As a further technical solution, the health product comprises a capsule, oral liquid.

[0020] Compared with the prior art, the present application has the following advantages:

[0021] The technical solution of the present application introduces low-temperature drying and pre-treatment by crushing, which effectively avoids the destruction of heat-sensitive antioxidant components (such as flavonoids and terpenoids) by high temperature, and at the same time, the particle size of the raw material is refined and the specific surface area is increased, providing a good material basis for subsequent extraction.

[0022] The low-temperature environment in the technical solution of the present application can inhibit the activity of oxidase and reduce the oxidative degradation of active ingredients; and moderate crushing can break the cell wall of the bark and release the essential oil components in the cells, thereby improving the subsequent extraction efficiency.

[0023] The present application adopts a three-stage supercritical carbon dioxide extraction process, which controls the temperature, pressure and time of supercritical carbon dioxide extraction in stages to realize efficient extraction and separation and purification of willow bark essential oil, and the yield is greatly improved compared with the traditional ethanol extraction method, and the content of active ingredients such as terpenes and flavonoids in the essential oil is significantly increased.

[0024] This is mainly due to the fact that supercritical carbon dioxide has high solubility of liquid and high diffusion coefficient of gas, and has selective solubility for polar and non-polar components under different pressure and temperature conditions. The low-temperature and low-pressure conditions in the early stage preferentially extract low-boiling volatile components (such as terpenes), the temperature and pressure are gradually increased in the middle stage to extract medium-polarity components (such as flavonoid aglycone), and the high-temperature and high-pressure conditions in the later stage extract high-boiling and slightly polar components (such as phenolic acids). The technical solution of the present application can avoid the residue of organic solvents, and the inert environment of carbon dioxide can prevent the oxidation of components, thereby maximizing the retention of the natural activity of essential oil.

[0025] The application evaluates the stability and antioxidant activity of the willow bark essential oil in the human digestive environment through simulated digestion and antioxidant activity evaluation, comprehensively evaluates the stability and antioxidant activity of the willow bark essential oil in the human digestive environment through simulated gastric juice and intestinal juice digestion experiments, combined with DPPH, ABTS, FRAP / T-AOC and other multi-index in vitro antioxidant evaluation and in vivo verification of oxidative stress animal models, and provides a scientific basis for its practical application. The simulated gastric juice digestion (pH 1.5-2.0, containing pepsin) can be used to investigate the stability of the components of the essential oil under acidic conditions, and the simulated intestinal juice digestion (pH 7.0-8.0, containing trypsin, bile salts and the like) can simulate the solubilization effect of bile salts on the liposoluble components in the alkaline environment. The experimental data show that the antioxidant activity of the essential oil extracted by the application only decreases by about 9% after gastric juice digestion, and the activity can recover to more than 99% of that before digestion in the later stage of intestinal juice digestion, which is closely related to the mechanism of bile salts promoting the release of liposoluble antioxidant components such as terpenes and flavonoids in the intestinal juice. The in vivo experiment confirms that the essential oil can significantly improve the oxidative stress state by scavenging free radicals and inhibiting lipid peroxidation through detecting the activities of SOD and GSH-Px and the content of MDA.

[0026] The application realizes the efficient extraction and activity retention of the willow bark essential oil through the innovative low-temperature drying-supercritical carbon dioxide extraction process, and comprehensively shows the antioxidant mechanism of the essential oil by combining the multi-dimensional antioxidant activity evaluation system. The technical scheme of the application not only solves the problems of component loss and activity reduction in the traditional extraction method, but also provides reliable theoretical support for the application of the essential oil in the fields of cosmetics, health products, food and the like through simulated digestion experiments and in vivo activity verification. From the perspective of resource utilization, the application fully develops the rich natural resource of willow bark, and the process has the characteristics of green environmental protection and high efficiency and controllability, which has important significance for promoting the comprehensive utilization and deep processing of willow resources and improving the added value of local characteristic products. The antioxidant activity of the willow bark essential oil extracted by the technology is significant, the clearance rate can reach more than 90% in the DPPH and ABTS free radical scavenging experiments, and the essential oil shows good stability and bioavailability in the simulated human digestive environment, which greatly widens the application field of the essential oil. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Metabolite distribution map in the bark of shrub willow 2345. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0029] The application provides an extraction process of willow bark essential oil and an evaluation method of antioxidant activity of the willow bark essential oil.

[0030] (I) the extraction process of the willow bark essential oil

[0031] Raw material pretreatment: the bark of shrub willows (such as Boshijian willow, Erliu willow and 2345 varieties) or arbor willows (such as Weiliu willow, Hanliu willow and 1011 varieties) is selected, impurities on the surface are removed, and then the bark is placed in a drying equipment and dried at a low temperature under the condition of 45-48 DEG C until the water content of the bark is less than 8%. The dried bark is crushed to 40-60 meshes and sieved for use.

[0032] Supercritical carbon dioxide extraction: the extraction is performed by using a supercritical carbon dioxide extraction device, and the specific extraction conditions are as follows:

[0033] The first stage: the extraction temperature is 30-33 DEG C, the extraction pressure is 33-35 MPa, the separation pressure is 10-12 MPa, and the extraction time is 45-48 minutes.

[0034] The middle stage: the extraction temperature is 33-35 DEG C, the extraction pressure is 35-38 MPa, the separation pressure is 8-10 MPa, and the extraction time is 75-78 minutes.

[0035] The last stage: the extraction temperature is 33.5-35 DEG C, the extraction pressure is 40-42 MPa, the separation pressure is 6-8 MPa, and the extraction time is 95-100 minutes.

[0036] Essential oil collection and purification: after the extraction is completed, the extraction liquid is collected, carbon dioxide is removed by reduced pressure distillation, and the crude willow bark essential oil is obtained, and then the purified willow bark essential oil is obtained by filtering through a 0.22 mu m filter membrane.

[0037] The analysis method of the components of the willow bark essential oil:

[0038] Determination of the physicochemical indexes of the willow bark essential oil

[0039] The contents of flavones, polyphenols, triterpenes, soluble sugars, soluble proteins and crude fats in the willow bark essential oils of different varieties are determined.

[0040] a. Determination of the content of flavones

[0041] The determination of the flavones is performed by using an aluminum chloride colorimetric method. 1g of the sample to be measured is placed in a triangular flask, 10mL of 95% ethanol solution is added, the flask is sealed and placed in a 50 DEG C constant temperature water bath for ultrasonic-assisted extraction for 0.5h, and then the filtrate is collected by filtering while hot. 1.0mL of the extraction liquid is accurately transferred into a 50mL volumetric flask, 2mL of aluminum chloride solution and 2.0mL of potassium acetate solution are sequentially added, the mixture is fully mixed, and then the volume is adjusted with 30% ethanol solution, the mixture is mixed, and then the absorbance is determined at 415nm within 30min by using a spectrophotometer. The calculation formula is as follows:

[0042]

[0043] Wherein: C is the flavonoids content (μg) calculated according to the standard curve; N is the dilution multiple of sample solution; VT is the total volume of extract (mL); VS is the sample volume (mL) taken for determination; m is the sample weight (g).

[0044] b. Determination of polyphenol content

[0045] The polyphenol content was determined by Folin-phenol colorimetry. 0.2 g of sample was weighed and transferred into a 10 mL centrifuge tube with 5 mL of distilled water, and then extracted in a 100°C water bath for 30 min. After cooling, the sample was diluted and filtered to obtain the filtrate. 0.1 mL of the filtrate was taken into a calibrated test tube, and 1 mL of Folin-phenol reagent and 3 mL of 7.5% sodium carbonate solution were added. The volume was made up to 10 mL with distilled water, and then mixed thoroughly. The color was developed at room temperature for 30-60 min in the dark, and then the absorbance was measured at 765 nm by a spectrophotometer. The calculation formula is as follows:

[0046]

[0047] Wherein: C is the polyphenol content (μg) calculated according to the standard curve; N is the dilution multiple of sample solution; VT is the total volume of extract (mL); VS is the sample volume (mL) taken for determination; m is the sample weight (g).

[0048] c. Determination of triterpenoids content

[0049] The triterpenoids content was determined by vanillin-glacial acetic acid colorimetry. 1 g of dry sample was weighed, and then 95% ethanol was added according to the solid-liquid ratio of 1:9. The sample was extracted in a 80°C water bath for 2-3 h. The supernatant was collected by centrifugation, and then the residue was extracted with 10 mL of 95% ethanol for 30 min. The supernatants were combined and diluted to 25 mL. 0.25 mL of ursolic acid standard solution was accurately taken, and then dried in a boiling water bath. Then, 5% vanillin-glacial acetic acid (0.4 mL) and perchloric acid (1.6 mL) were added in sequence. The mixture was heated in a 65°C water bath for 45 min, and then 5 mL of glacial acetic acid was added after the reaction was cooled to room temperature. The mixture was shaken and placed at room temperature for 15 min. The absorbance was measured at 548 nm by a spectrophotometer. The calculation formula is as follows:

[0050]

[0051] Wherein: C is the triterpenoids content (mg) calculated according to the standard curve; VT is the total volume of extract (mL); VS is the sample volume (mL) taken for determination; m is the sample weight (g).

[0052] d. Determination of soluble sugar content

[0053] The soluble sugar content was determined by anthrone colorimetry. The experimental operation was divided into two parts: standard curve establishment and sample analysis. The standard curve construction process was as follows: 100 μg / mL sucrose stock solution was prepared by gradient dilution to prepare 10-100 μg / mL of 6 standard solutions. Precisely pipette 1 mL of each concentration solution (1 mL of ultrapure water for the blank group) into a 25 mL ground test tube, quantitatively add 5 mL of anthrone color developing agent, vortex well, then react in a boiling water bath for 10 minutes, and quickly transfer to a flowing water bath for cooling for 20 minutes. Use a UV-visible spectrophotometer to detect the absorbance at 630 nm wavelength, and establish a concentration-absorbance linear regression equation. Sample processing procedure: accurately weigh 0.20 g of seed powder, add 10 mL of deionized water, and extract in boiling water for 30 minutes for three times, combine the extraction solution, purify it through a filter membrane, and then dilute to 25 mL in a volumetric flask. Mix 0.1 mL of the sample to be tested with 1.9 mL of ultrapure water, then add 0.5 mL of anthrone-ethyl acetate complex reagent and 5 mL of concentrated sulfuric acid, shake well, and then treat in a boiling water bath for 60 seconds. After cooling at room temperature, measure the absorbance at 630 nm wavelength. Set three replicates for each sample, and calculate the soluble sugar content according to the following formula based on the standard curve:

[0054]

[0055] In the formula: C is the soluble sugar content obtained in the standard curve, μg; a is the volume of the sample solution, mL; V is the volume of the extraction solution, mL; n is the dilution multiple; and W is the weight of the seed sample, g.

[0056] e. Determination of soluble protein content

[0057] The soluble protein content was determined by the Coomassie brilliant blue method. 0.2 g of seed sample was weighed and added to 8 mL of Tris-HCl buffer (0.02 M, pH 7.5) to grind into homogenate (2 mL of buffer was added first, and the remaining 6 mL was washed in three times). The homogenate was transferred to a 10 mL centrifuge tube and centrifuged at 7000 x g for 20 min. The supernatant was taken and mixed with 5 mL of Coomassie brilliant blue G-250 reagent. The absorbance (OD value) was measured at 595 nm with a blank as a control. The test was repeated three times. The soluble protein content was calculated according to the following formula:

[0058]

[0059] In the formula: C is the standard curve value, μg; V is the total volume of the extraction solution, mL; W is the fresh weight of the sample, g; and VS is the sample amount added when measured, mL.

[0060] f. Determination of crude fat content and acid phosphatase activity

[0061] Fat content was determined using Soxhlet extraction method. 0.2 g of seed sample was wrapped in a defatted filter paper and placed in a Soxhlet extractor, petroleum ether was added, and the extraction was performed continuously for 12 hours at 40°C constant temperature water bath. After the extraction was completed, the filter paper was transferred to a 105°C oven for drying to remove residual petroleum ether, then placed in a desiccator to cool to room temperature, and weighed accurately after cooling. The crude fat content was calculated according to the following formula:

[0062]

[0063] W1 - filter paper weight, g; W2 - dry weight of seed sample, g; W3 - extracted sample weight, g.

[0064] 3. Determination of metabolite composition in willow bark essential oil

[0065] Metabolite extraction

[0066] Sample preparation: The dried willow bark was taken out from the -80°C freezer, thawed, and vortexed to mix.

[0067] Freeze-drying: An appropriate amount of sample was placed in a numbered centrifuge tube, frozen at -80°C overnight, and vacuum freeze-dried.

[0068] Extraction: 70% methanol containing internal standard extraction solution was added at a concentration of 30 times, vortexed, ice water bath ultrasonic, and then centrifuged.

[0069] Filtering and storage: The supernatant was removed and filtered with a 0.22 μm filter membrane, then stored in a sample bottle for testing.

[0070] Mass spectrometry data acquisition and analysis

[0071] Instrument system: UPLC and MS / MS system were used.

[0072] Liquid phase conditions:

[0073] Chromatographic column: Agilent SB-C18 1.8 μm.

[0074] Mobile phase: A phase ultrapure water (0.1% formic acid), B phase acetonitrile (0.1% formic acid).

[0075] Elution gradient: 0-9 min B phase 5%-95%, maintain for 1 min, 10-11.1 min decrease to 5%, balance to 14 min.

[0076] Flow rate: 0.35 mL / min; column temperature: 40°C; sample size: 2 μL.

[0077] Mass spectrometry conditions: ESI 500°C, IS voltage ±5500V, gas settings GSI 50 psi, GSII 60 psi, CUR 25 psi, high CID, MRM mode, optimized DP and CE.

[0078] Qualitative and quantitative analysis of substances:

[0079] Qualitative analysis: Self-built database, based on secondary spectrum information, remove isotopes, ion duplicate signals and fragment ions.

[0080] Quantitation: MRM mode, screening precursor ions, selecting characteristic fragment ions for accurate quantification, and exporting data after integral correction.

[0081] Data processing

[0082] Software processing: Analyst 1.6.3 was used to screen characteristic ions, MultiQuant was used for integration and correction, and peak area data were exported.

[0083] ④ Determination of flavonoid targeted metabolome in willow bark essential oil

[0084] Metabolite extraction

[0085] Vacuum freeze-dried willow bark essential oil.

[0086] Grind into powder using a ball mill at a frequency of 30 Hz for 1.5 minutes.

[0087] Weigh 20 mg of powder and add 10 μL of internal standard mixture (4000 nmol / L) and 500 μL of methanol (70%).

[0088] After ultrasonic treatment for 30 minutes, centrifugation was performed at 4°C (12000 r / min, 5 minutes), and the supernatant was collected and filtered through a 0.22 μm filter membrane. The supernatant was stored in an injection bottle for LC-MS / MS analysis.

[0089] Mass spectrometry data acquisition and analysis

[0090] Instrument system: UPLC (ExionLC TM AD) and MS / MS( 6500+).

[0091] Liquid phase conditions: chromatographic column Waters ACQUITY HS ST3C18 (1.8 μm, 100 mm × 2.1 mm), mobile phase A (ultrapure water + 0.05% formic acid), B (acetonitrile + 0.05% formic acid), flow rate 0.35 mL / min, column temperature 40 ° C, injection volume 2 μL, elution gradient changed according to the preset ratio.

[0092] Mass spectrometry conditions: ESI temperature 550°C, voltage ±5500 V in positive / negative ion mode, CUR 35 psi, optimized DP and CE, Q-Trap 6500+ scanning detection.

[0093] Qualitative and quantitative: qualitative based on standard database, quantitative by MRM mode, calculate the content of sample by standard curve after integration.

[0094] Data processing

[0095] Software processing: Analyst 1.6.3 and MultiQuant 3.0.3 process mass spectrum data.

[0096] Standard curve: prepare standard solution with different concentrations, draw standard curve.

[0097] Content calculation: substitute the peak area ratio of sample into the standard curve equation, calculate the content of flavonoids in sample (nmol / g), formula: c*V / 1000000 / m (c is sample concentration, V is the volume of extract, m is sample mass).

[0098] 5. Determination of flavor electronic nose and volatile metabolome of willow bark essential oil

[0099] Electronic nose detection:

[0100] Take an appropriate amount of sample, put it into the electronic nose, start reading and record.

[0101] Metabolite extraction and mass spectrum detection

[0102] Metabolite extraction:

[0103] Take out the sample and grind it in liquid nitrogen, vortex mix, take 500 mg (or 1 mL liquid) into the headspace bottle.

[0104] Add saturated NaCl solution and internal standard solution, full-automatic headspace solid phase microextraction (HS-SPME).

[0105] Mass spectrum data acquisition and analysis:

[0106] HS-SPME conditions: oscillation at 60℃ for 5 minutes, headspace extraction of extraction head for 15 minutes, desorption at 250℃ for 5 minutes.

[0107] Chromatographic conditions: DB-5MS column, high-purity helium flow rate 1.2 mL / min, programmed temperature to 280℃ for 5 minutes.

[0108] Mass spectrum conditions: EI source, selected ion detection mode (SIM), accurate scan qualitative and quantitative ions.

[0109] Data processing: MassHunter software processes data, self-built database for qualitative and quantitative analysis, internal standard semi-quantitative method to calculate the relative content of VOCs.

[0110] Relative content calculation formula:

[0111] Solid sample: Xi = (Vs x Cs) / M x Ii / Is x 10 -3

[0112] Liquid sample: Xi = (Vs x Cs) / V x Ii / Is x 10 -3

[0113] (Xi is the content of the compound, Vs is the volume of the internal standard, Cs is the concentration of the internal standard, M / V is the mass / volume of the sample, Ii / Is is the peak area of the analyte / internal standard)

[0114] Targeted hormone assay of willow bark essential oil

[0115] Based on the UHPLC-MS-MS platform, the content changes of endogenous salicylic acid (SA), methyl salicylate (MeSA), cinnamic acid, o-hydroxycinnamic acid, and salicylic acid-2-O-β-glucoside were qualitatively and quantitatively determined by targeted metabolomics. The extraction solvent was chromatographically pure acetonitrile, the chromatographic separation system was composed of methanol-0.1% formic acid (phase A) and water-0.1% formic acid (phase B), and the analysis platform was configured with a Shimadzu LC-30AD ultra-high performance liquid chromatography system coupled with a triple quadrupole mass spectrometer (SCIEX 6500+), a low-temperature high-speed centrifuge, and a vacuum concentration device.

[0116] 0.2 g of sample was weighed, 2 mL of pre-cooled acetonitrile was added, and extraction was carried out at 4°C in the dark for 16 h, and the supernatant was collected by centrifugation at 12000 x g for 10 min. The extraction was repeated twice, and the organic phase was combined and purified by a C18 solid phase extraction column. After nitrogen blowing to dryness, 200 μL of methanol was used for redissolution, and the sample was purified by a 0.22 μm organic microporous filter membrane. An Agilent Poroshell 120 EC-C18 reversed-phase column (2.1 x 150 mm, 2.7 μm) was used, and the mobile phase gradient program was set as follows: 0-1 min, 20% A; 1-3 min, linearly increased to 50% A; 3-9 min, gradient to 80% A; 9-10.5 min, maintained at 80% A; 10.5-10.6 min, dropped to 20% A; 10.6-13.5 min, system balance. The flow rate was constant at 0.3 mL / min, the column temperature was 30°C, and the injection volume was 2 μL. An electrospray ionization source (ESI ±) and a multiple reaction monitoring mode (MRM) were used, and typical ion pairs included gibberellin GA1 (Q1347.0→Q3259.3), auxin IAA (Q1176.2→Q3130.0), etc. Auxiliary parameters: gas curtain gas pressure 15 psi, ion source temperature 400°C, atomization gas and auxiliary gas flow rate optimized to the best signal-to-noise ratio. Gradient standards of 0.5-50 μg / mL were prepared, and the hormone content was calculated according to the formula:

[0117]

[0118] Wherein: C - detection concentration, ng / mL; V - extraction volume, mL; m - sample mass, g.

[0119] (II) Evaluation method of antioxidant activity of willow bark essential oil

[0120] 1. Evaluation of in vitro antioxidant activity

[0121] a. Simulated gastric juice digestion: mix the extracted willow bark essential oil solution with simulated gastric juice (papain: 2000 U / mL (Sigma-Aldrich P7000 type), hydrochloric acid concentration: 0.1 mol / L, pH: 1.5-2.0, temperature / time: 37°C / 2h) in a certain proportion, adjust the pH to 1.5-2.0, and incubate in a 37°C constant temperature water bath for 2h, sample every 0.5h, and measure the antioxidant activity (FRAP / T-AOC, DPPH, ABTS) to observe the changes in the gastric juice; in the simulated gastric juice digestion, the volume ratio of willow bark essential oil solution to simulated gastric juice is 1:5, and the concentration of the essential oil solution is 1mg / mL.

[0122] b. Simulated intestinal juice digestion: adjust the pH of the sample after gastric juice digestion to 7.0-8.0 with 1 mol / L NaOH solution, add simulated intestinal juice (trypsin: 10 mg / mL (Solarbio T8000 type, activity ≥ 250 U / mg), bile salt: 10 g / L (sodium cholate, Sigma-Aldrich B8631 type), α-amylase: 5 U / mL (Sigma-Aldrich A3176 type), lipase: 1 U / mL (Sigma-Aldrich L3126 type), pH: 7.0-8.0, temperature / time: 37°C / 3h), continue to incubate in a 37°C constant temperature water bath for 3h, sample every 0.5h, and measure the antioxidant activity (FRAP / T-AOC, DPPH, ABTS) to analyze the digestion characteristics in the intestinal juice;

[0123] In the simulated intestinal juice digestion, the volume ratio of the sample after gastric juice digestion to simulated intestinal juice is 1:3;

[0124] DPPH free radical scavenging experiment: according to the method of Appendix D in GB / T 23776-2018 "Sensory Evaluation Methods for Tea", the purified willow bark essential oil is prepared into a series of concentration solutions of 0.1-10 mg / mL with ethanol. Take 2 mL of essential oil solution and mix with 2 mL of 0.06-0.6 mmol / L DPPH ethanol solution, avoid light for 30-40 minutes, and measure the absorbance at 517 nm wavelength. Calculate the DPPH free radical scavenging rate, the formula is as follows:

[0125]

[0126] Wherein, A0 is the absorbance of the mixture of DPPH solution and ethanol, A1 is the absorbance of the mixture of essential oil solution and DPPH solution, and A2 is the absorbance of the mixture of essential oil solution and ethanol.

[0127] ABTS free radical scavenging assay: Following the method in Appendix C of GB5009.268-2016, "National Food Safety Standard - Determination of Multiple Elements in Food," mix equal volumes of 7 mM ABTS solution and 2.45 mM potassium persulfate solution. Incubate in the dark for 12 hours to obtain the ABTS free radical working solution. Dilute with phosphate buffer (pH 7.4) before use to an absorbance of 0.7 ± 0.02 at 734 nm. Mix 1 mL of the essential oil solution with 4 mL of the ABTS working solution, react for 6 minutes, and measure the absorbance at 734 nm.

[0128] The ABTS free radical scavenging rate was calculated using the following formula:

[0129]

[0130] Where A0 is the absorbance of ABTS working solution, and A1 is the absorbance of the mixture of essential oil solution and ABTS working solution.

[0131] 2. Evaluation of Antioxidant Activity in Vivo

[0132] Establishment of an Oxidative Stress Animal Model and Grouping: Male Kunming mice, 6-8 weeks old, weighing 20±2g, were randomly divided into five groups, with 10 mice in each group: a blank control group, a model control group, and a low-dose (50 mg / kg), a medium-dose (100 mg / kg), and a high-dose (200 mg / kg) willow bark essential oil group. The oxidative stress model was established in the model control and essential oil-treated groups by intraperitoneal injection of D-galactose (120 mg / kg) once daily for 6 weeks. The blank control group received an equal volume of normal saline.

[0133] Administration: Starting from the first day of modeling, the mice in the essential oil treatment group were gavaged with the corresponding dose of willow bark essential oil (dissolved in corn oil) every day, while the blank control group and the model control group were gavaged with the same volume of corn oil.

[0134] Index detection: After the experiment, mice were killed and liver tissue was quickly removed. Superoxide dismutase (SOD) activity (according to the WST-1 method, GB / T5009.171-2003), glutathione peroxidase (GSH-Px) activity (according to the enzyme-linked immunosorbent assay, GB / T5009.152-2003), and malondialdehyde (MDA) content (according to the thiobarbituric acid method, GB / T5009.181-2003) in liver tissue were measured using kits from the Nanjing Jiancheng Bioengineering Institute.

[0135] (III) Application of Willow Bark Essential Oil

[0136] The extracted willow bark essential oil can be used to prepare antioxidant products, including but not limited to:

[0137] Cosmetics: face masks, facial cleansers, soaps, shampoos, etc., with an addition of 0.1-5% (mass fraction).

[0138] Health products: capsules, oral liquids, etc., with a recommended daily intake of 50-200 mg.

[0139] Food: as a natural antioxidant added to edible oils, baked goods, beverages, etc., with an addition of 0.01-0.5% (mass fraction).

[0140] The following are specific examples

[0141] Example 1: Extraction of Weeping Willow Bark Essential Oil (45°C Drying, Standard Extraction Conditions)

[0142] Raw material pretreatment: Fresh weeping willow bark was selected, surface impurities were removed, and it was dried at 45°C to a moisture content of 6.5%, and then crushed to 40 mesh.

[0143] Supercritical extraction:

[0144] Early stage: temperature 30°C, pressure 33 MPa, separation pressure 10 MPa, extraction time 45 minutes.

[0145] Middle stage: temperature 33°C, pressure 35 MPa, separation pressure 8 MPa, extraction time 75 minutes.

[0146] Late stage: temperature 33.5°C, pressure 40 MPa, separation pressure 6 MPa, extraction time 95 minutes.

[0147] Essential oil yield: 3.25% (based on the dry weight of the bark).

[0148] Example 2: Extraction of Willow 2345 Variety Bark Essential Oil (46°C Drying, Standard Extraction Conditions)

[0149] Raw material pretreatment: Dry willow bark was selected, impurities were removed, and it was dried at 46°C to a moisture content of 5.8%, and then crushed to 50 mesh.

[0150] Supercritical extraction:

[0151] Early stage: temperature 31°C, pressure 34 MPa, separation pressure 11 MPa, extraction time 46 minutes.

[0152] Middle stage: temperature 34°C, pressure 36 MPa, separation pressure 9 MPa, extraction time 76 minutes.

[0153] Late stage: temperature 34°C, pressure 41 MPa, separation pressure 7 MPa, extraction 96 minutes.

[0154] Essential oil yield: 3.52%.

[0155] Example 3: Extraction of 1011 variety bark essential oil (47°C drying, standard extraction conditions)

[0156] Raw material pretreatment: 1011 variety bark dried at 47°C to 7.2% moisture content, ground to 60 mesh.

[0157] Supercritical extraction:

[0158] Early stage: temperature 32°C, pressure 35 MPa, separation pressure 12 MPa, extraction 47 minutes.

[0159] Middle stage: temperature 35°C, pressure 37 MPa, separation pressure 10 MPa, extraction 77 minutes.

[0160] Late stage: temperature 34.5°C, pressure 42 MPa, separation pressure 8 MPa, extraction 97 minutes.

[0161] Essential oil yield: 3.48%.

[0162] Example 4: Extraction of Weiji willow bark essential oil (48°C drying, standard extraction conditions)

[0163] Raw material pretreatment: Weiji willow bark dried at 48°C to 6.1% moisture content, ground to 40 mesh.

[0164] Supercritical extraction:

[0165] Early stage: temperature 33°C, pressure 33 MPa, separation pressure 10 MPa, extraction 48 minutes.

[0166] Middle stage: temperature 33°C, pressure 35 MPa, separation pressure 8 MPa, extraction 78 minutes.

[0167] Late stage: temperature 35°C, pressure 40 MPa, separation pressure 6 MPa, extraction 98 minutes.

[0168] Essential oil yield: 3.61%.

[0169] Example 5: Extraction of Erliu willow bark essential oil (46°C drying, optimized extraction conditions)

[0170] Raw material pretreatment: Erliu willow bark dried at 46°C to 6.3% moisture content, ground to 50 mesh.

[0171] Supercritical extraction:

[0172] Pre-stage: temperature 32°C, pressure 34 MPa, separation pressure 11 MPa, extraction for 47 minutes.

[0173] Mid-stage: temperature 34°C, pressure 37 MPa, separation pressure 9 MPa, extraction for 77 minutes.

[0174] Post-stage: temperature 35°C, pressure 41 MPa, separation pressure 7 MPa, extraction for 99 minutes.

[0175] Essential oil yield: 3.75%.

[0176] Comparative Example 1: Conventional ethanol extraction method (using willow bark as an example)

[0177] Raw material pretreatment: Weeping willow bark was dried at 45°C and ground to 40 mesh.

[0178] Extraction process: Ethanol reflux extraction method was used, with a solid-liquid ratio of 1:10 (g / mL), an ethanol concentration of 70%, a temperature of 70°C, a reflux time of 3 hours, and repeated extraction twice.

[0179] Comparative Example 2: High-temperature drying extraction (using willow bark as an example)

[0180] Raw material pretreatment: The willow bark was dried at 60°C to a moisture content of 5.5% and ground to 50 mesh.

[0181] Supercritical extraction: The extraction conditions were the same as those of Example 2.

[0182] Test

[0183] (I) In vitro antioxidant activity experiment

[0184] The essential oils extracted from willow bark in Examples 1-5 and Comparative Examples 1-2 were prepared into 1 mg / mL ethanol solutions, and the DPPH and ABTS free radical scavenging experiments were performed according to the above methods, with each sample repeated 3 times and the average value taken.

[0185] Table 1

[0186]

[0187]

[0188] As can be seen from Table 1, the in vitro experiment: the DPPH and ABTS radical scavenging rates of the willow bark essential oil of Examples 1-5 are significantly higher than those of Comparative Example 1 and Comparative Example 2 (P < 0.05), among which the scavenging rate of Example 5 is the highest, indicating that the extraction process of the present application can effectively retain the antioxidant components in the essential oil. Comparative Example 1 uses traditional ethanol extraction method, due to the action of high temperature and organic solvent, part of the antioxidant components are inactivated or degraded, the scavenging rate is low; Comparative Example 2 uses high temperature drying, which destroys the heat-sensitive antioxidant components in the bark, thereby reducing the antioxidant activity of the essential oil.

[0189] Antioxidant activity experiment after simulated digestion

[0190] Select Example 5 (two willow bark essential oil) and Comparative Example 1 (traditional ethanol extraction essential oil), according to the above simulated gastric juice and intestinal juice digestion steps, sample at 0h (before digestion), 0.5h, 1h, 1.5h, 2h (gastric juice digestion) and 2.5h, 3h, 3.5h, 4h, 5h (intestinal juice digestion) respectively, and determine the DPPH and ABTS radical scavenging rate;

[0191] Table 2

[0192]

[0193]

[0194]

[0195] As can be seen from Table 2, in the gastric juice digestion stage: the DPPH and ABTS scavenging rates of Example 5 decrease slightly with time (about 9% and 9.2% from 0h to 2h), indicating that part of the antioxidant components are slightly degraded under acidic conditions, but the overall stability is good; the scavenging rate of Comparative Example 1 decreases by about 13.5% and 15.8%, indicating that the components of the essential oil extracted by traditional method are more susceptible to inactivation in gastric juice.

[0196] In the intestinal juice digestion stage: the scavenging rate of Example 5 gradually recovers and approaches the level before digestion (the DPPH and ABTS scavenging rates at 5h are restored to 99.8% and 99.6% respectively), which may be related to the bile salts in intestinal juice promoting the release of lipid-soluble antioxidant components; the scavenging rate of Comparative Example 1 only recovers to 90.9% and 89.5% of the pre-digestion level, showing that the components thereof are more significantly lost during digestion.

[0197] (II) In vivo antioxidant activity experiment

[0198] Select Example 5 (two willow bark essential oil) and Comparative Example 1 (traditional ethanol extraction essential oil), and perform experiments according to the above in vivo antioxidant activity evaluation method, and detect the SOD, GSH-Px activity and MDA content in the liver tissue of each group of mice.

[0199] Table 3

[0200]

[0201]

[0202]

[0203] As can be seen from Table 3, in the in vivo experiment: compared with the model control group, the SOD and GSH-Px activities in the liver tissue of the mice in the high-dose group of Example 5 were significantly increased (P<0.05), and the MDA content was significantly reduced (P<0.05), close to the level of the blank control group; although the indicators of the Comparative Example 1 group were improved to a certain extent, the effect was not as obvious as that of the Example 5 group. This shows that the extracted willow bark essential oil has good in vivo antioxidant effect and can effectively alleviate the damage of oxidative stress to the body.

[0204] In summary, the willow bark essential oil extraction process provided by the present application can efficiently extract essential oil with excellent antioxidant activity by optimizing the drying temperature and supercritical extraction conditions, thereby providing reliable technical support for the development and utilization of willow bark resources.

[0205] Through non-targeted determination of metabolites in the bark of shrub willow 2345, the top five main metabolites are: ketones, aldehydes and esters account for 16.75%, terpenoids account for 16.56%, flavonoids account for 6.16%, organic acids account for 5.69%, and polyphenols account for 4.14%, and the specific distribution is as shown in Figure 1 .

[0206] The preferred embodiments of the present application disclosed above are only used to help explain the present application. The preferred embodiments do not describe all the details, nor limit the present application to the specific embodiments described. Obviously, many modifications and changes can be made according to the content of the present application.

Claims

1. A process for extracting willow bark essential oil, characterized in that: The following steps are involved: The willow bark is dried at a low temperature of 45-48°C and then crushed to 40-60 mesh. It is extracted by supercritical carbon dioxide extraction. The extraction conditions are: front temperature 30-33°C, front pressure 33-35MPa, separation pressure 10-12MPa, extraction for 45-48 minutes; medium temperature 33-35°C, medium pressure 35-38MPa, separation pressure 8-10MPa, extraction for 75-78 minutes; post-temperature 33.5-35°C, post-pressure 40-42MPa, separation pressure 6-8MPa, and post-stage extraction for 95-100 minutes.

2. The extraction process according to claim 1, characterized in that The willow bark includes shrub willow and tree willow.

3. The extraction process according to claim 2, characterized in that The tree willows include weeping willows, matsudanax, and 1011 varieties, and the shrub willows include dustpan willows, two willows, and 2345 varieties. Impurities on the bark surface need to be removed before drying.

4. The method for evaluating the antioxidant activity of the willow bark essential oil obtained by the process according to claim 1, 2 or 3, wherein: include: In vitro evaluation included simulated gastric and intestinal digestion to determine DPPH free radical scavenging and ABTS free radical scavenging; In vivo evaluation, an animal model of oxidative stress was established, and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) content in the liver tissue of the animal model were detected.

5. The evaluation method according to claim 4, wherein: The DPPH free radical scavenging comprises: The essential oil was prepared into a 0.1-10 mg / mL ethanol solution, mixed with a 0.06-0.6 mmol / LDPPH ethanol solution, reacted in the dark for 30-40 minutes, and the absorbance was measured at 517 nm.

6. The evaluation method according to claim 4, wherein: The ABTS free radical scavenging comprises: 7 mM A ABTS solution was mixed with 2.45 mM potassium persulfate in the dark for 12 h, diluted and reacted with the essential oil solution for 6 min, and the absorbance was measured at 734 nm.

7. The evaluation method according to claim 4, wherein: The in vivo oxidative stress animal model is established by intraperitoneal injection of D-galactose, and antioxidant indicators are detected after oral administration of willow bark essential oil.

8. Use of the willow bark essential oil obtained according to any one of claims 1 to 3 in the preparation of antioxidant products, characterized in that: The antioxidant products include: cosmetics, health products, and food.

9. The use according to claim 8, characterized in that The cosmetics include facial masks, facial cleansers, soaps, and shampoos.

10. The use according to claim 8, characterized in that The health care products include capsules and oral liquids.

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