CN113075016A - Extraction process and activity determination method for ultrasonically-assisted flash extraction of osmanthus hydrolat - Google Patents
Extraction process and activity determination method for ultrasonically-assisted flash extraction of osmanthus hydrolat Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4022—Concentrating samples by thermal techniques; Phase changes
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4022—Concentrating samples by thermal techniques; Phase changes
- G01N2001/4033—Concentrating samples by thermal techniques; Phase changes sample concentrated on a cold spot, e.g. condensation or distillation
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Abstract
The invention discloses an extraction process and an activity determination method for ultrasonically-assisted flash extraction of osmanthus hydrolat. Soaking the flower of the osmanthus with saline solution, carrying out ultrasonic treatment after carrying out flash crushing, adding water for direct distillation, and inoculating the osmanthus hydrolat; the sweet osmanthus hydrolat is aromatic water with strong sweet osmanthus fragrance and terpene components, is prominent in sweet after aging and placing, and has peach-like pleasant fruit sweet. According to the invention, through the strong breaking force of the flash extractor, the cavitation generated between the rotary vane and the liquid, and the ultrasonic wave breaks the plant cell wall, more active substances in the petals are brought into the solution, and the additional value of the osmanthus fragrans product is improved; and provides in vitro and cell whitening experiments, an antioxidant determination method and experimental data of the osmanthus hydrolat, and proves that the osmanthus hydrolat has extremely low cytotoxicity and antioxidant activity and whitening activity.
Description
Technical Field
The invention belongs to the field of daily chemical products, relates to osmanthus hydrolat, and particularly relates to an extraction process for ultrasonically-assisted flash extraction of osmanthus hydrolat, a determination method for antioxidant and whitening activity and basic data.
Background
Osmanthus fragrans (Osmanthus fragrans) is a evergreen shrub or a small arbor in Oleaceae, one of ten traditional famous flowers in China. The osmanthus fragrans is pleasant in flower fragrance, overflows when being thick, is clean and dustless, can be eaten as a medicine, and has more than 2500 years of medicinal and edible history in China. The sweet osmanthus is warm and pungent in flavor, warms stomach and relieves pain, can reduce phlegm and dissipate blood stasis, and has curative effects on inappetence, decayed tooth resistance, amenorrhea and abdominal pain; sweet osmanthus essential oil, extract and absolute oil are favored in the fields of flavor blending and aromatherapy. In recent years, the by-product of aromatic plant distillation, hydrolat, is more and more popular with consumers; various plant hydrolat products (including osmanthus hydrolat) appear on the market as skin lotion, and the effects of diminishing inflammation, whitening, removing yellow odor, removing acne, resisting aging and the like are declared to be endless. In fact these effects benefit from the rather complex mixture in the hydrolat, containing minor amounts of essential oils and various water-soluble components. However, the activity of the hydrolat is mostly studied on bacteriostasis, and other functional cognition is mostly recorded in an ancient book, and practical experience and even analogy are inferred, so that research on the activity and safety evaluation of the hydrolat needs to provide an applicable determination method.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: how to evaluate the activity and safety of the osmanthus hydrolat and how to fully utilize the osmanthus plant resources so as to enrich more active ingredients in the extraction link of the osmanthus hydrolat.
In order to solve the above-mentioned plausible problems, the invention provides an extraction process for ultrasonically-assisted flash extraction of osmanthus hydrolat, which comprises the following steps:
step 1): soaking the dried sweet osmanthus in salt solution;
step 2): carrying out flash extraction on the saline solution soaked with the sweet osmanthus flowers obtained in the step 1) on a flash extractor;
step 3): uniformly stirring the extract obtained in the step 2), and putting the extract in an ultrasonic instrument for ultrasonic treatment;
step 4): standing the material obtained in the step 3), putting the material into a sterilized distillation device for distillation, and adding distilled water; timing with boiling as a standard starting point, controlling the distillation rate, and taking the steam condensate by using a sterilization bottle;
step 5): naturally cooling the condensate obtained in the step 4) to room temperature in a super clean bench, filtering in a sterile environment, sealing the obtained pure dew, and refrigerating.
Preferably, the setting voltage of flash extraction in the step 2) is 15V; the ultrasonic power in the step 3) is 600W, and the ultrasonic time is not less than 30 min.
The invention also provides a method for measuring the antioxidant activity of the osmanthus hydrolat, which is characterized in that the osmanthus hydrolat prepared by the ultrasonic-assisted flash extraction process of the osmanthus hydrolat is diluted into different concentrations to be measured according to a double dilution method; DPPH free radical scavenging experiment and ABTS free radical scavenging experiment were performed separately.
Preferably, the DPPH radical scavenging experiment specifically comprises: each sample was mixed with 0.16mM DPPH solution diluted with absolute ethanol in a 96-well plate and left at 30 ℃ in the dark; after 30 minutes, their absorbance at 517nm was measured by using a microplate reader; vitamin C was used as a positive control and ethanol was used as a blank control, each experiment was repeated three times, the results were averaged, and the DPPH free radical scavenging formula was as follows:
％inhibition＝(1-A1/A0)×100％；
wherein A is1Absorbance of the sample and DPPH solution, A0Absorbance of absolute ethanol and DPPH solution.
Preferably, the ABTS free radical scavenging experiment is specifically as follows: fully mixing 7.0mmol/L ABTS aqueous solution and 4.90mmol/L potassium persulfate aqueous solution according to the volume ratio of 1:1, placing the mixed solution at the dark room temperature for overnight reaction to obtain dark green solution containing ABTS cation free radicals, diluting the dark green solution with absolute ethyl alcohol, and taking the solution as working solution when the absorbance measured by a spectrophotometer is 0.700 +/-0.020 nm; and (3) mixing the sample and the working solution in each concentration in a volume ratio of 1:1, sequentially adding the materials into a 96-well plate, and storing the materials in the dark at the temperature of 30 ℃; after 6min, measuring the absorbance at 734nm by using an enzyme-labeling instrument, taking vitamin C as a positive control and ethanol as a blank control, and calculating the ABTS free radical clearance rate according to the following formula:
％inhibition＝(1-B1/B0)×100％
wherein, B1Is the absorbance of the sample and ABTS working solution, B0Absorbance of absolute ethanol and ABTS working solution.
The invention also provides an experimental method for inhibiting the activity of the tyrosinase in vitro of the osmanthus hydrolat, which is characterized in that the osmanthus hydrolat prepared by the extraction process for ultrasonically-assisted flash extraction of the osmanthus hydrolat is diluted into different concentrations to be tested according to a double dilution method; the pH of the buffer solution PBS is 6.8, the tyrosinase solution is 50U/mL, and the L-DOPA solution is 5 mM; first, sequentially adding PBS into A, B, C, D wells; respectively adding samples into A, B holes, then putting tyrosinase solution into A and C, after reaction, finally adding L-DOPA solution into four holes for continuous incubation for 5 minutes, measuring absorbance at 475nm, and selecting kojic acid as a positive control; the in vitro tyrosinase activity inhibition rate calculation formula is as follows:
wherein, A is the absorbance of the tyrosinase solution and the sample, B is the absorbance of the tyrosinase solution and the sample, C is the absorbance of the tyrosinase solution and the sample, and D is the absorbance of the tyrosinase solution or the sample.
The invention also provides a method for measuring the cytotoxicity and the whitening activity of the osmanthus hydrolat, which comprises a cell proliferation detection experiment, an intracellular tyrosinase activity measurement experiment and an intracellular melanin content experiment of the osmanthus hydrolat; firstly, setting different concentration gradients as a sample group for the osmanthus hydrolat prepared by the ultrasonic-assisted flash extraction process of the osmanthus hydrolat; then setting a negative control group, a blank control group and a positive control group;
the cell proliferation detection experiment specifically comprises the following steps: taking B16F10 cells in logarithmic growth phase, and performing cell selection according to 4X 103The cells/well were plated in 96-well plates and medium containing 10% CCK8, 5% CO was added at 100. mu.L/well2Culturing in a constant-temperature incubator at 37 ℃; detecting the absorbance value at 450nm by using an enzyme-labeling instrument; the formula for calculating relative vitality is as follows:
relative activity% (sample group OD value-blank OD value)/(negative control group OD value mean value-blank OD value) × 100;
the test for measuring the activity of the intracellular tyrosinase specifically comprises the following steps: counting B16F10 cells in logarithmic growth phase, adjusting cell concentration, and selecting at 4 × 103Individual cells/well were seeded into 96-well plates in 5% CO2Culturing overnight in a constant-temperature incubator at 37 ℃; adding samples with various concentrations and positive control kojic acid solution at 100 μ L/well, setting 3 multiple wells for each treatment, and culturing for 48 h; removing the culture medium, adding 50 μ L of aqueous solution of Triton-X100 with mass percent of 1% into each well, quickly freezing in a refrigerator at-80 deg.C for 30min, taking out, and thawing in water bath at 37 deg.C to completely break cells; adding 100 mu L of 1mg/mL L-DOPA solution, carrying out water bath at 37 ℃ for 40min, and detecting the OD value of each hole under 480nm by using an enzyme-labeling instrument; the formula for tyrosinase activity is as follows:
tyrosinase activity ═ 100% (test well OD-blank well OD)/(control well OD-blank well OD) ×
The experiment of the melanin content in the cells specifically comprises the following steps: counting B16F10 cells in logarithmic growth phase, adjusting cell concentration, and selecting at 4 × 103Individual cells/well were seeded into 96-well plates in 5% CO2Culturing overnight in a constant-temperature incubator at 37 ℃; adding samples with various concentrations and positive control kojic acid solution at 100 μ L/well, setting 3 multiple wells for each treatment, and culturing for 48 h; removing the culture medium, adding 100 μ L of 1mol/L NaOH solution containing 10% DMSO, performing water bath at 80 deg.C for 1h to completely dissolve melanin, and measuring absorbance of each well at wavelength of 405nm with microplate reader; the calculation formula of the relative content of melanin is as follows:
relative melanin content (test OD-blank OD)/(control OD-blank OD) × 100%
Preferably, the positive control group adopts 10mg/mL kojic acid.
According to the invention, through the strong breaking force of the flash extractor, the cavitation generated between the rotary vane and the liquid, and the ultrasonic wave breaks the plant cell wall, more active substances in the petals are brought into the solution, and the additional value of the osmanthus fragrans product is improved; and provides in vitro and cell whitening experiments, an antioxidant determination method and experimental data of the osmanthus hydrolat, and proves that the osmanthus hydrolat has extremely low cytotoxicity and antioxidant activity and whitening activity.
Drawings
FIG. 1 is a flow diagram of total ion flow of constituents of Osmanthus fragrans dew;
FIG. 2 shows the oxidation resistance of Osmanthus fragrans lour hydrosol;
FIG. 3 shows the tyrosinase activity inhibition rate in Osmanthus fragrans dew;
FIG. 4 is the effect of Osmanthus fragrans hydrolat on cell proliferation;
FIG. 5 is the effect of Osmanthus fragrans hydrolat on the activity of cellular tyrosinase;
FIG. 6 shows the effect of Osmanthus fragrans lour hydrolat on the melanin content of cells.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.
Example 1
Extracting the osmanthus hydrolat by an ultrasonic-assisted flash distillation method:
1. experimental equipment and drugs:
3000mL beaker, 5000mL flask, spherical condenser, Clevenger device, glass rod, glass bead, sterile bottle,
flash extractor (JBHT-50T, Henan Zhi Jing Biotech limited), ultrasonic cell pulverization (Scientz-IID, Ningbo Xinzhi Biotech limited, China), sterile low-speed filter paper, super clean bench; electronic balance model PL203 (mettler-toledo instruments (shanghai) ltd).
2. Extracting the osmanthus hydrolat:
1) soaking 100g of osmanthus flowers which are rapidly dried at 70 ℃ in 1000mL of salt water;
2) carrying out flash extraction on the material obtained in the step 1) for 30s at a voltage of 15V;
3) after stirring evenly from top to bottom and from left to right, the materials are placed in an ultrasonic instrument to be ultrasonically treated for 3 times with 600W power, and each time lasts for 10 minutes.
4) Standing the material obtained in the step 3) for 11.5 hours, putting the material into a sterilized distillation device for distillation, and adding 1000mL of distilled water; the distillation rate was controlled at about 5.1mL/min, timed with boiling as the standard starting point, and 3h of steam condensate was taken using a sterile bottle.
5) And (3) naturally cooling the condensate obtained in the step (4) to room temperature in a super clean bench, filtering at low speed in a sterile environment, taking out a small amount of the condensate to be tested, sealing the residual hydrolat, and then putting the condensate into a refrigerator at 5-8 ℃ for refrigeration.
Example 2
Determining the antioxidant activity of the osmanthus hydrolat:
1. experimental equipment and drugs:
ultraviolet spectrophotometer UV-6000 type (Shanghai chromatography instruments, Inc.); microplate reader (Multiskan FC, Thermo Fisher Technology Co., Ltd., China); a 96-well plate; anhydrous ethanol, 1-diphenyl-2-picrylhydrazino (free radical) (DPPH), 2-diaza-bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS), potassium persulfate, disodium hydrogen phosphate, and sodium dihydrogen phosphate (shanghai tai taktac technologies, ltd.).
2. The experimental steps are as follows:
the hydrolat was diluted to the following sample concentrations according to the double dilution method:
q (pure sample), 0.5Q, 0.25Q, 0.125Q, 0.0625Q;
1) DPPH free radical scavenging experiment
Each sample and a 0.16mM DPPH solution (diluted with absolute ethanol) were mixed in a 96-well plate and left at 30 ℃ in the dark. After 30 minutes, their absorbance at 517nm was measured by using a microplate reader. Vitamin C was used as a positive control and ethanol was used as a blank control. Each set of experiments was repeated three times and the results averaged.
The DPPH radical scavenging formula is as follows:
％inhibition＝(1-A1/A0)×100％
A1: sample and DPThe absorbance of the PH solution is measured by the PH sensor,
A0: absorbance of absolute ethanol and DPPH solution.
2) ABTS free radical scavenging experiments
The ABTS cation free radical is obtained by oxidizing ABTS through sulfate. An aqueous ABTS solution (7.0mM) and an appropriate amount of an aqueous potassium persulfate solution (4.90mM) are mixed well according to a volume ratio of 1:1, and then the mixture is placed in the dark at room temperature for overnight reaction to obtain a dark green solution containing ABTS cationic radicals. Diluting with anhydrous ethanol, and taking the solution as working solution when the absorbance measured by a spectrophotometer is 0.700 +/-0.020 nm. The samples and working solution at each concentration were added to a 96-well plate in a volume ratio of 1:1 in sequence and stored in the dark at 30 ℃. After 6min, the absorbance was measured at 734nm with a microplate reader. Vitamin C is used as a positive control, and ethanol is used as a blank control. The ABTS free radical clearance is calculated according to the following formula:
％inhibition＝(1-B1/B0)×100％
B1: the absorbance of the sample and the ABTS working solution,
B0: absorbance of absolute ethanol and ABTS working solution.
Example 3
In-vitro tyrosinase activity inhibition experiment of osmanthus hydrolat
1. Experimental equipment and drugs:
ultraviolet spectrophotometer UV-6000 type (Shanghai chromatography instruments, Inc.); microplate reader (Multiskan FC, Thermo Fisher Technology Co., Ltd., China); a constant-temperature water bath kettle, an ultrasonic cleaning instrument, a liquid-transferring gun, a volumetric flask, a 96-hole plate and a centrifuge tube; tyrosinase, derived from mushroom (Shanghai Michelle chemical technology Co., Ltd.), kojic acid, PBS (pH 6.8), L-DOPA, absolute ethanol (Shanghai Tantake technology Co., Ltd.), ultrapure water.
2. The experimental steps are as follows:
to A, B, C, D wells, 100 μ L, 150 μ L, and 200 μ L of PBS (pH 6.8) were sequentially added. mu.L of each sample was added to each of the A and B wells, and then 50. mu.L of tyrosinase solution (50U/mL) was put into the A and C wells, and reacted at 37 ℃ for 10 minutes. A solution of 100. mu. L L-DOPA (5mM) was added rapidly to the four wells. After incubating the 96-well plate at 37 ℃ for 5 minutes continuously, absorbance was measured at 475 nm. Kojic acid was selected as a positive control. The in vitro tyrosinase activity inhibition rate calculation formula is as follows:
a is the absorbance of the existing tyrosinase solution and the sample, B is the absorbance of the existing sample but not the tyrosinase solution, C is the absorbance of the existing tyrosinase solution and not the sample, and D is the absorbance of the nonexistence tyrosinase solution or the sample.
Example 4
Cell proliferation experiment of osmanthus hydrolat
1. Experimental equipment and drugs:
CO2 constant temperature incubator (Beijing Mulberry next laboratory research institute, model WIGGENSWCI-180); an ultra-clean bench (Shanghai Du Te scientific instruments Co., Ltd., model: SW-CJ-2 FD); a centrifuge (Shanghai Luxiang apparatus centrifuge Co., Ltd., model: TD 5); microplate reader (TECAN, model: SPARK 10M) fetal bovine serum (Biological Industries, cat: 04-007-1A); DMEM (NaHCO3, 1.5g/L) medium (Saururi David: iCell-128-0001); PBS (Procell, cat # WH 0112201911 XP); DMEM medium: the fetal serum was prepared in a ratio of 9:1 in a cell complete medium, pancreatin (Biosharp, cat # 143188); CCK8 (Invigenech, cat # IV 08-100); B16F10 cells, purchased from pith of shanghai.
2. The experimental steps are as follows:
1) first, the samples were divided into Sample groups, Sample (concentration 16%, 4%, 1%, 0.25%, 0.0625%), negative control group, blank control group and positive control (10mg/mL kojic acid) for 3 groups.
2) Counting B16F10 cells in logarithmic growth phase, adjusting cell concentration, and selecting at 4 × 103Individual cells/well were seeded into 96-well plates in 5% CO2(CO in the gas volume in the incubator)25% in the same manner as below), and culturing at a constant temperature of 37 deg.CThe culture was carried out overnight in a chamber.
3) Adding each gradient sample and positive control kojic acid solution at 100 μ L/well, setting 3 multiple wells for each treatment, washing each well with PBS three times, adding culture medium containing 10% CCK8, 5% CO at 100 μ L/well2And culturing in a constant temperature incubator at 37 ℃ for 2 hours. And detecting the absorbance value at 450nm by using a microplate reader. Relative activity% (% OD value-blank OD value of sample group)/(OD value mean value-blank OD value of negative control group) × 100
Example 5
Test for measuring intracellular tyrosinase activity of osmanthus hydrolat
1. Experimental equipment and drugs:
CO2a constant temperature incubator (Peking Mulbernext laboratory research institute, model: WIGGENSWCI-180); an ultra-clean bench (Shanghai Du Te scientific instruments Co., Ltd., model: SW-CJ-2 FD); a centrifuge (Shanghai Luxiang apparatus centrifuge Co., Ltd., model: TD 5); microplate reader (TECAN, model: SPARK 10M) fetal bovine serum (Biological Industries, cat: 04-007-1A); DMEM (NaHCO)31.5g/L) medium (sibilant grove bio-lot number: iCell-128-0001); DMEM medium: preparing a cell complete culture medium, PBS (Procell, cat # WH 0112201911 XP) with a fetal serum ratio of 9: 1; pancreatin (Biosharp, cat # 143188); triton X-100(Solarbio, cat # T8200)
2. The experimental steps are as follows:
1) first, the samples were divided into Sample groups, Sample (concentration 16%, 8%, 4%, 2%, 1%), negative control group, blank control group and positive control (10mg/mL kojic acid) for 3 groups.
2) Counting B16F10 cells in logarithmic growth phase, adjusting cell concentration, and selecting at 4 × 103Individual cells/well were seeded into 96-well plates in 5% CO2And culturing overnight in a constant temperature incubator at 37 ℃. Samples of various concentrations and positive control kojic acid solution were added at 100. mu.L/well, 3 duplicate wells were set for each treatment, and incubated for 48 h.
3) Removing the culture medium, washing each well twice with PBS, adding 50 μ L of 1% Triton-X100 water solution into each well, quickly freezing in a refrigerator at-80 deg.C for 30min, taking out, and thawing in water bath at 37 deg.C for about 30min to completely break the cells.
4) Adding 100 mu L of 1mg/mL L-DOPA solution, carrying out water bath at 37 ℃ for 40min, and detecting the OD value of each well under 480nm by using an enzyme-labeling instrument.
Tyrosinase activity ═ 100% (test well OD-blank well OD)/(control well OD-blank well OD) ×
Example 6
Experiment of intracellular melanin content of osmanthus hydrolat
1. Experimental equipment and drugs:
same as example 4
2. The experimental steps are as follows:
1) first, the samples were divided into Sample groups, Sample (concentration 16%, 8%, 4%, 2%, 1%), negative control group, blank control group and positive control (10mg/mL kojic acid) for 3 groups.
2) Counting B16F10 cells in logarithmic growth phase, adjusting cell concentration, and selecting at 4 × 103Individual cells/well were seeded into 96-well plates in 5% CO2And culturing overnight in a constant temperature incubator at 37 ℃. Samples of various concentrations and positive control kojic acid solution were added at 100. mu.L/well, 3 duplicate wells were set for each treatment, and incubated for 48 h.
3) The medium was removed. Each well was washed twice with PBS, 100. mu.L of a 1mol/L NaOH solution containing 10% DMSO was added, the melanin was completely dissolved in a water bath at 80 ℃ for 1 hour, and the absorbance of each well at a wavelength of 405nm was measured with a microplate reader.
Relative melanin content (test OD-blank OD)/(control OD-blank OD) × 100%
The specific data results for each example are as follows:
TABLE 1 Osmanthus fragrans hydrolat principal ingredients and relative contents
TABLE 2 Oxidation resistance of Osmanthus hydrolat
TABLE 3 Osmanthus fragrans hydrolat in vitro tyrosinase activity inhibition rate
TABLE 4 Effect of Osmanthus hydrolat on cell proliferation
TABLE 5 Effect of Osmanthus hydrolat on cellular tyrosinase Activity
TABLE 6 influence of Osmanthus hydrolat on the content of melanin in cells
In conclusion, the invention provides the process for extracting the osmanthus hydrolat by the ultrasonic-assisted flash distillation method, the osmanthus hydrolat with elegant fragrance and sufficient fruit feeling is obtained, and meanwhile, a determination scheme and data support are provided for proving the functionality and safety of the osmanthus hydrolat.
Claims (8)
1. An extraction process for ultrasonically assisting flash extraction of osmanthus hydrolat is characterized by comprising the following steps:
step 1): soaking the dried sweet osmanthus in salt solution;
step 2): carrying out flash extraction on the saline solution soaked with the sweet osmanthus flowers obtained in the step 1) on a flash extractor;
step 3): uniformly stirring the extract obtained in the step 2), and putting the extract in an ultrasonic instrument for ultrasonic treatment;
step 4): standing the material obtained in the step 3), putting the material into a sterilized distillation device for distillation, and adding distilled water; timing with boiling as a standard starting point, controlling the distillation rate, and taking the steam condensate by using a sterilization bottle;
step 5): naturally cooling the condensate obtained in the step 4) to room temperature in a super clean bench, filtering in a sterile environment, sealing the obtained pure dew, and refrigerating.
2. The ultrasonic-assisted flash extraction process of osmanthus hydrolat according to claim 1, wherein the set voltage of flash extraction in the step 2) is 15V; the ultrasonic power in the step 3) is 600W, and the ultrasonic time is not less than 30 min.
3. The determination method for the antioxidant activity of the osmanthus hydrolat is characterized in that the osmanthus hydrolat prepared by the ultrasonic-assisted flash extraction process of the osmanthus hydrolat of claim 1 or 2 is diluted into different concentrations to be measured according to a double dilution method; DPPH free radical scavenging experiment and ABTS free radical scavenging experiment were performed separately.
4. The determination method for the antioxidant activity of the osmanthus hydrolat according to claim 3, wherein the DPPH free radical scavenging experiment specifically comprises: each sample was mixed with 0.16mM DPPH solution diluted with absolute ethanol in a 96-well plate and left at 30 ℃ in the dark; after 30 minutes, their absorbance at 517nm was measured by using a microplate reader; vitamin C was used as a positive control and ethanol was used as a blank control, each experiment was repeated three times, the results were averaged, and the DPPH free radical scavenging formula was as follows:
％inhibition＝(1-A1/A0)×100％；
wherein A is1Absorbance of the sample and DPPH solution, A0Absorbance of absolute ethanol and DPPH solution.
5. The determination method for the antioxidant activity of the osmanthus hydrolat according to claim 3, wherein the ABTS free radical scavenging experiment specifically comprises the following steps: fully mixing 7.0mmol/L ABTS aqueous solution and 4.90mmol/L potassium persulfate aqueous solution according to the volume ratio of 1:1, placing the mixed solution at the dark room temperature for overnight reaction to obtain dark green solution containing ABTS cation free radicals, diluting the dark green solution with absolute ethyl alcohol, and taking the solution as working solution when the absorbance measured by a spectrophotometer is 0.700 +/-0.020 nm; and (3) mixing the sample and the working solution in each concentration in a volume ratio of 1:1, sequentially adding the materials into a 96-well plate, and storing the materials in the dark at the temperature of 30 ℃; after 6min, measuring the absorbance at 734nm by using an enzyme-labeling instrument, taking vitamin C as a positive control and ethanol as a blank control, and calculating the ABTS free radical clearance rate according to the following formula:
％inhibition＝(1-B1/B0)×100％
wherein, B1Is the absorbance of the sample and ABTS working solution, B0Absorbance of absolute ethanol and ABTS working solution.
6. An in vitro tyrosinase activity inhibition experiment method of osmanthus hydrolat is characterized in that the osmanthus hydrolat prepared by the ultrasonic-assisted flash extraction technology of the osmanthus hydrolat in claim 1 or 2 is diluted into different concentrations to be tested according to a double dilution method; the pH of the buffer solution PBS is 6.8, the tyrosinase solution is 50U/mL, and the L-DOPA solution is 5 mM; first, sequentially adding PBS into A, B, C, D wells; respectively adding samples into A, B holes, then putting tyrosinase solution into A and C, after reaction, finally adding L-DOPA solution into four holes for continuous incubation for 5 minutes, measuring absorbance at 475nm, and selecting kojic acid as a positive control; the in vitro tyrosinase activity inhibition rate calculation formula is as follows:
wherein, A is the absorbance of the tyrosinase solution and the sample, B is the absorbance of the tyrosinase solution and the sample, C is the absorbance of the tyrosinase solution and the sample, and D is the absorbance of the tyrosinase solution or the sample.
7. A method for measuring cytotoxicity and whitening activity of osmanthus hydrolat is characterized by comprising a cell proliferation detection experiment, an intracellular tyrosinase activity measurement experiment and an intracellular melanin content experiment of the osmanthus hydrolat; firstly, setting different concentration gradients by taking the osmanthus hydrolat prepared by the ultrasonic-assisted flash extraction technology of the osmanthus hydrolat of claim 1 or 2 as a sample group; then setting a negative control group, a blank control group and a positive control group;
the cell proliferation detection experiment specifically comprises the following steps: taking B16F10 cells in logarithmic growth phase, and performing cell selection according to 4X 103The cells/well were plated in 96-well plates and medium containing 10% CCK8, 5% CO was added at 100. mu.L/well2Culturing in a constant-temperature incubator at 37 ℃; detecting the absorbance value at 450nm by using an enzyme-labeling instrument; the formula for calculating relative vitality is as follows:
relative activity% (sample group OD value-blank OD value)/(negative control group OD value mean value-blank OD value) × 100;
the test for measuring the activity of the intracellular tyrosinase specifically comprises the following steps: counting B16F10 cells in logarithmic growth phase, adjusting cell concentration, and selecting at 4 × 103Individual cells/well were seeded into 96-well plates in 5% CO2Culturing overnight in a constant-temperature incubator at 37 ℃; adding samples with various concentrations and positive control kojic acid solution at 100 μ L/well, setting 3 multiple wells for each treatment, and culturing for 48 h; removing the culture medium, adding 50 μ L of aqueous solution of Triton-X100 with mass percent of 1% into each well, quickly freezing in a refrigerator at-80 deg.C for 30min, taking out, and thawing in water bath at 37 deg.C to completely break cells; adding 100 mu L of 1mg/mL L-DOPA solution, carrying out water bath at 37 ℃ for 40min, and detecting the OD value of each hole under 480nm by using an enzyme-labeling instrument; the formula for tyrosinase activity is as follows:
tyrosinase activity ═ 100% (test well OD-blank well OD)/(control well OD-blank well OD) ×
The experiment of the melanin content in the cells specifically comprises the following steps: counting B16F10 cells in logarithmic growth phase, adjusting cell concentration, and selecting at 4 × 103Individual cells/well were seeded into 96-well plates in 5% CO2Culturing overnight in a constant-temperature incubator at 37 ℃; adding samples with various concentrations and positive control kojic acid solution at 100 μ L/well, setting 3 multiple wells for each treatment, and culturing for 48 h; removing the culture medium, adding 100 μ L of 1mol/L NaOH solution containing 10% DMSO, performing water bath at 80 deg.C for 1h to completely dissolve melanin, and measuring absorbance of each well at wavelength of 405nm with microplate reader; the calculation formula of the relative content of melanin is as follows:
relative melanin content (test OD-blank OD)/(control OD-blank OD) × 100%.
8. The method for measuring the cytotoxicity and the whitening activity of the osmanthus hydrolat according to claim 7, wherein 10mg/mL kojic acid is adopted in the positive control group.
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| CN115715750A (en) * | 2021-09-30 | 2023-02-28 | 太和康美(北京)中医研究院有限公司 | Osmanthus fragrans flower extract, preparation method and skin care application thereof |
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| CN114306163A (en) * | 2022-01-10 | 2022-04-12 | 山东花物堂生物科技有限公司 | Preparation method and application of bidens pilosa extract with whitening effect |
| CN116370386A (en) * | 2023-05-31 | 2023-07-04 | 广州巴宝莉化妆品有限公司 | Method for extracting active stock solution of fresh flowers and cosmetics |
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Application publication date: 20210706 |