Preparation method of saffron or chicory oil-soluble extract

Abstract

The present application relates to a kind of preparation methods of safflower or chicory oil-soluble extract, comprising the following steps: (1) the safflower or chicory raw material is soaked with water, then freeze-thaw is carried out multiple times, to obtain liquor;(2) to the liquor, add vegetable oil stirring extraction, after centrifugal demulsification, oil phase is separated;The oil phase is added to another liquor, again stirring extraction;Again centrifugal demulsification, oil phase is separated, add drying agent to remove residual water, filter, obtain oil-soluble extract.The present application is repeatedly freeze-thaw to destroy the structure of plant cell, so that intracellular material is released, remove lipophilic solvent penetration plant cell barrier, so that oil-soluble component can be completely contacted with lipophilic solvent, improve yield, with good market application prospect.

Description

Technical Field

[0001] The invention belongs to the field of plant extraction, and particularly relates to a method for preparing a saffron or chicory oil-soluble extract. Background Art

[0002] Saffron (Crocus sativus L.), also known as saffron, is the upper part of the style and stigma of the crocus flower, a plant of the Iridaceae family. It is considered the world's most expensive spice. Traditional Chinese Medicine believes that saffron is sweet and mild in nature, and has the effects of promoting blood circulation, harmonizing qi and blood, dispersing blood stasis, and calming the mind. Modern pharmacological research has found that saffron can alleviate atherosclerosis and promote fat metabolism, thereby lowering blood lipids. Saffron also has an antioxidant effect by inhibiting oxygen free radicals. Saffron also has the benefits of promoting blood circulation, removing blood stasis, unblocking meridians, cooling blood and detoxifying, relieving inflammation and pain, enhancing immunity, detoxifying and beautifying the skin, promoting sleep, and increasing energy.

[0003] Chicory (Cichorium intybus), also known as blue chrysanthemum, is slightly bitter, salty and cool in nature. It has the effects of clearing the liver and promoting bile secretion, strengthening the stomach and digestion, and promoting diuresis and reducing swelling. It is used to treat damp-heat jaundice, stomach pain, poor appetite, edema and oliguria.

[0004] Currently, saffron and chicory are extracted using water or alcohol, and further research into the pharmacological activity of these extracts is ongoing. There are no reports on the use of oils to extract saffron and chicory. The aqueous and alcoholic extracts of saffron and chicory primarily contain polysaccharides, flavonoids, and saponins, which, when used in skincare products, suffer from poor transdermal absorption. Extracting saffron's oil-soluble components using supercritical carbon dioxide is expensive.

[0005] Besides supercritical carbon dioxide extraction, there are three main methods for preparing plant oil-soluble extracts: 1. Steam distillation, such as the method reported in CN102100719. 2. Extracting the plant material with water or alcohol, recovering the solvent to obtain an extract, then adding the extract to a lipophilic solvent, dissolving and filtering to obtain an oil-soluble extract (the method reported in CN100551265). 3. Extracting the plant material directly in a matrix oil or lipophilic solvent, such as the methods reported in CN109846795 and CN102028060.

[0006] Saffron has a variety of pharmacological activities that are beneficial to human skin. The most important mechanism of biological effect of saffron (C. sativus) is antioxidant and anti-inflammatory activity. The antioxidant stress of saffron is related to the activation of nuclear factor-erythroid factor 2-related factor 2 (NRF2). NRF2 is the master transcription factor of endogenous antioxidant. When NRF2 is activated, the antioxidant enzymes that autonomously scavenge free radicals in the cell, including downstream glutathione, SOD, and CAT enzyme systems, are also upregulated. These enzymes can directly scavenge ROS in the mitochondria. Free radicals damage the skin in many ways, including DNA damage, protein denaturation, and lipid oxidation. Therefore, skin antioxidant function can be said to be the foundation of skin health. Promoting NRF2 is a precise and effective antioxidant mechanism.

[0007] The existing technology has the following problems:

[0008] (1) Supercritical extraction is expensive and not conducive to industrial production.

[0009] (2) Steam distillation can only extract oil-soluble components with low boiling points, and the yield of oil-soluble components with high boiling points is low. Heat-sensitive components are easily destroyed.

[0010] (3) The method of first extracting with water or alcohol and then dissolving the extract with oil has a low yield of effective ingredients because only a small amount of oil-soluble components can be extracted by extraction with water or alcohol.

[0011] (4) Oil or lipophilic solvents have weak penetration into plant cells, and direct extraction with oil or lipophilic solvents results in a low yield of the target component. Summary of the Invention

[0012] The technical problem to be solved by the present invention is to provide a method for preparing an oil-soluble extract of saffron or chicory. The method destroys the structure of plant cells through repeated freezing and thawing, releases intracellular substances, removes obstacles that prevent lipophilic solvents from penetrating plant cells, allows oil-soluble components to completely contact with the lipophilic solvent, improves the yield, and has good market application prospects.

[0013] The present invention provides a method for preparing an oil-soluble extract of saffron or chicory, comprising the following steps:

[0014] (1) soaking saffron or chicory raw materials in water, and then freezing and thawing them multiple times to obtain a liquid feed;

[0015] (2) Adding vegetable oil to the feed liquid and stirring for extraction, separating the oil phase after centrifugal demulsification; adding the oil phase to another feed liquid and stirring and extracting again; centrifuging again to demulsify and separate the oil phase, adding a desiccant to remove residual water, filtering, and obtaining an oil-soluble extract.

[0016] The safflower or chicory raw material in step (1) is crushed to obtain safflower or chicory powder with a mesh size of 40-60.

[0017] The freeze-thawing in step (1) is freezing at-18 to-80℃ for 6-24h and thawing at 50-60℃.

[0018] The plant oil in step (2) is one or more of sunflower oil, peanut oil, corn oil, soybean oil, rapeseed oil, and palm oil.

[0019] The stirring extraction temperature in step (2) is 50-60℃, and the stirring extraction time is 1-2h.

[0020] The centrifugal demulsification rotation speed in step (2) is 6000-8000rpm, and the centrifugal demulsification time is 20-30min.

[0021] The drying agent in step (2) is one or more of anhydrous calcium sulfate, anhydrous calcium chloride, anhydrous sodium sulfate, and molecular sieves.

[0022] Beneficial effects

[0023] (1) The present application can destroy the structure of plant cells by repeated freeze-thawing, release intracellular substances, remove the obstacles for penetration of lipophilic solvents into plant cells, and make the oil-soluble components fully contact with the lipophilic solvents, thereby improving the yield and having good market application prospect.

[0024] (2) In the present application, the lipophilic solvent is contacted with the plant material liquid subjected to multiple freeze-thawing, which can greatly improve the concentration of the target components in the oil phase.

[0025] (3) The extraction temperature of the present application is not high, which can avoid the destruction of heat-sensitive components; compared with conventional alcohol or water extracts, the transdermal absorption capacity is stronger; and the final extract is dissolved in oil, and the extract is more stable and less likely to discolor due to the low oxygen solubility in oil. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The color change of the extract of Example 2 after being placed in an environment of 45℃ for 3 weeks.

[0027] Figure 2 The color change of the extract of Comparative Example 5 after being placed in an environment of 45℃ for 3 weeks.

[0028] Figure 3 The effect of UV irradiation (10mJ / cm 2 ) and safflower oil-soluble extract (0.1%, 0.2%, 0.5%) treatment for 24h on the gene expression of HaCat cells.

[0029] Figure 4 UV irradiation (10mJ / cm 2 ) and saffron oil-soluble extract (0.5%) were treated for 24 h to evaluate the changes in CAT, SOD1, and SOD2 expression in HaCat cells (*VS NC group, #VS UV group). DETAILED DESCRIPTION

[0030] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0031] Example 1

[0032] 1. Freezing and breaking the cell wall

[0033] Crush 100g of saffron stamens, pass through a 40-mesh sieve, and soak in 500g of pure water (5 times the amount of the plant material) for 2 hours. Freeze the material to -20°C for 6 hours. Thaw the frozen material in a 50-60°C water bath. Once completely thawed, freeze it again at -20°C for 6 hours, then thaw it in a 50-60°C water bath.

[0034] 2. Medium temperature extraction

[0035] Add 500 g of vegetable oil to the liquid in step 1, and stir and extract at 50-60° C. for 2 h. The vegetable oil includes, but is not limited to, sunflower oil, peanut oil, corn oil, soybean oil, rapeseed oil, and palm oil; in this embodiment, sunflower oil is selected.

[0036] 3. Demulsification

[0037] The mixture from step 2 was centrifuged at 8000 rpm for 20 min to separate the oil phase and the aqueous phase containing the plant residue (the aqueous phase was mixed with new plant oil and subjected to extraction in step 2).

[0038] 4. Extract again at medium temperature

[0039] The oil phase in step 3 was added to the freshly frozen-thawed liquid and extracted with stirring at 50-60°C for 2h.

[0040] 5. Demulsification

[0041] The mixture in step 4 was centrifuged at 8000 rpm for 20 min to separate the oil phase. A desiccant was added to the oil phase, stirred for 4 h, allowed to stand for 12 h, and filtered to obtain the saffron oil-soluble extract. The desiccant was selected from anhydrous calcium sulfate, anhydrous calcium chloride, anhydrous sodium sulfate, and molecular sieves; in this embodiment, anhydrous calcium sulfate was selected.

[0042] Example 2

[0043] 1. Freezing and breaking the cell wall

[0044] Take 100g of chicory root, crush it, pass it through a 60-mesh sieve, add 5 times the amount of pure water (500g) as the plant material, and soak it for 2 hours. Freeze the material to -20°C for 12 hours. Thaw the frozen material in a 50-60°C water bath. Once completely thawed, freeze it again at -20°C for 12 hours, then thaw it in a 50-60°C water bath.

[0045] 2. Medium temperature extraction

[0046] Add 500 g of vegetable oil to the liquid in step 1, and stir and extract at 50-60° C. for 2 h. The vegetable oil includes, but is not limited to, sunflower oil, peanut oil, corn oil, soybean oil, rapeseed oil, and palm oil; peanut oil is selected in this embodiment.

[0047] 3. Demulsification

[0048] The mixture from step 2 was centrifuged at 8000 rpm for 20 min to separate the oil phase and the aqueous phase containing the plant residue (the aqueous phase was mixed with new plant oil and subjected to extraction in step 2).

[0049] 4. Extract again at medium temperature

[0050] The oil phase in step 3 was added to the freshly frozen-thawed liquid, and the mixture was stirred and extracted at 50-60°C for 1-2 hours.

[0051] 5. Demulsification

[0052] The mixture from step 4 is centrifuged at 8000 rpm for 20 minutes to separate the oil phase. A desiccant is added to the oil phase, stirred for 4 hours, allowed to stand for 12 hours, and filtered to obtain the chicory oil-soluble extract. The desiccant is selected from anhydrous calcium sulfate, anhydrous calcium chloride, anhydrous sodium sulfate, and molecular sieves; in this embodiment, anhydrous calcium chloride is selected.

[0053] Comparative Example 1

[0054] This comparative example provides a preparation method of saffron extract. Compared with Example 1, the difference is that 100g of saffron is crushed, passed through a 40-mesh sieve, and then directly extracted with 500g of 50-60℃ vegetable oil for 2h, filtered, and the extract is obtained.

[0055] Comparative Example 2

[0056] This comparative example provides a method for preparing a saffron extract. 100g of saffron was ground, passed through a 40-mesh sieve, and extracted with 500g of 50% ethanol under reflux for 2 hours. The extract was recovered to obtain a solid powder. 500g of vegetable oil was added to the solid powder, stirred at 50-60°C for 2 hours, filtered, and the filtrate was collected to obtain the saffron oil-soluble extract.

[0057] Comparative Example 3

[0058] This comparative example provides a method for preparing chicory extract. Compared with Example 2, the difference is that 100g of chicory root is crushed, passed through a 60-mesh sieve, and then directly extracted with 500g of 50-60°C vegetable oil for 2h, and filtered to obtain the chicory extract.

[0059] Comparative Example 4

[0060] This comparative example provides a method for preparing a chicory extract. 100g of chicory root was ground, passed through a 60-mesh sieve, and extracted with 500g of 50% ethanol under reflux for 2 hours. The extract was recovered to obtain a solid powder. 500g of vegetable oil was added to the solid powder, stirred at 50-60°C for 2 hours, filtered, and the filtrate was collected to obtain the chicory oil-soluble extract.

[0061] Comparative Example 5

[0062] This comparative example provides a method for preparing chicory extract: 100g of chicory root is crushed, passed through a 60-mesh sieve, and extracted with 500g of 50% butanediol at 50-60°C for 2h with stirring, filtered, and the filtrate is collected to obtain the chicory extract.

[0063] Test Example 1 Determination of Safranal Content

[0064] 1. Preparation of Reference Solution

[0065] Accurately weigh 25.0 mg of saffron aldehyde reference substance, place it in a 10 mL volumetric flask, dissolve it in methanol and dilute to the scale as the reference solution for later use.

[0066] 2. Preparation of test solution

[0067] Measure 7 mL of saffron oil-soluble extract, place it in a 15 mL centrifuge tube, add 2 mL of methanol, shake for about 1 min, centrifuge at 5000 rpm for 10 min, take the supernatant, filter it with a 0.45 μm nylon membrane, and use it as the test solution for later use.

[0068] 3. Chromatographic conditions

[0069] Chromatographic column: Agilent SB-C18 (4.6 mm × 250 mm, 5 μm);

[0070] Mobile phase:

[0071] Time / min 0 40 41 46 Mobile phase A 10％ 100％ 20％ 10％ Mobile phase B 90％ 0％ 80％ 90％

[0072] Elution time: 46 min;

[0073] Flow rate: 0.8 mL / min;

[0074] Detection wavelength: 360nm;

[0075] Column temperature: 25°C;

[0076] 1, 2, 5, 10, 15, 20, and 30 μl of the reference solution were aspirated for liquid chromatography analysis. A standard curve was drawn with the amount of saffron aldehyde injected as the abscissa and the peak area of ​​saffron aldehyde as the ordinate. The content of saffron aldehyde in the test sample was calculated based on the standard curve.

[0077] Results: The contents of safranal in Example 1, Comparative Example 1, and Comparative Example 2 were 0.28%, 0.07%, and 0.11%, respectively, indicating that the technology of the present invention has obvious advantages.

[0078] Test Example 2 Determination of β-sitosterol content

[0079] 1. Preparation of reference solution

[0080] Accurately weigh 5.0 mg of β-sitosterol standard, place it in a 5 mL volumetric flask, add an appropriate amount of ethanol and ultrasonically dissolve it for 10 minutes. After cooling to room temperature, dilute to the scale and use it as the reference solution for later use.

[0081] 2. Preparation of test solution

[0082] Accurately measure 7 mL of chicory oil-soluble extract and place it in a 15 mL centrifuge tube. Add 2 mL of methanol and shake for about 1 min. Centrifuge at 5000 rpm for 10 min. Take the supernatant and filter it with a 0.45 μm filter membrane. Use it as the test solution for later use.

[0083] 3. Chromatographic conditions

[0084] Chromatographic column: Agilent Extend-C18 (4.6 mm × 250 mm, 5 μm);

[0085] Mobile phase: methanol

[0086] Elution time: 30 min;

[0087] Flow rate: 1.0 mL / min;

[0088] Detection wavelength: 210nm;

[0089] Column temperature: 35°C;

[0090] Pipette 2, 5, 10, 15, 20, 30, and 50 μL of the reference solution for liquid chromatography analysis. Plot a standard curve using the injected β-sitosterol volume as the abscissa and the β-sitosterol peak area as the ordinate. Calculate the β-sitosterol content in the test sample based on the standard curve.

[0091] Results: The β-sitosterol contents in Example 2, Comparative Example 3, and Comparative Example 4 were 0.47%, 0.09%, and 0.18%, respectively, indicating that the technology of the present invention is significantly advanced.

[0092] Test Example 3 Stability Investigation

[0093] like Figure 1 and 2 As shown, after the extracts of Example 2 and Comparative Example 5 were placed in a 45°C environment, the color of the sample of Example 2 did not change significantly after 3 weeks, while the color of the sample of Comparative Example 5 became significantly darker. This indicates that the stability of the oil-soluble extract is significantly better than that of the alcohol extract.

[0094] Experimental Example 4: Real-time quantitative PCR detection of antioxidant genes in saffron oil-soluble extracts

[0095] (1) Human keratinocytes (HaCat) were cultured in complete DMEM (containing 10% fetal bovine serum and 1% penicillin-streptomycin). When the cells reached 80% to 90% confluence, they were seeded in 96-well cell culture plates (4,000 cells). Culture was continued for 24 hours to allow the cells to fully adhere.

[0096] (2) Replace the culture medium with serum-free medium and culture for 6-12 hours. The experiment was divided into a control group and an experimental group. The control group was cultured with fresh culture medium, and the experimental group was cultured with culture medium containing different concentrations of active substances. The culture was continued for 24 hours. Then, 10 μL of CCK-8 solution was added to the well plate and the culture was continued for 1.5 hours. The cell viability after treatment with different concentrations of active substances was calculated using an enzyme-linked microplate reader according to the CCK-8 standard, and the maximum non-lethal concentration of the active substance was screened out.

[0097] (3) Cell culture and grouping: Using the optimal concentration screened, 24-well plates were used for cell seeding and active substance treatment. Three groups were set up: negative control group (NC), UV irradiation group (UV 10 mJ / cm 2 ), saffron essential oil test group. 3 replicates per group;

[0098] (4) mRNA level detection: Cell lysis, RNA extraction and Realtime-PCR were performed to detect NRF2 downstream antioxidant genes and evaluate the effects of active ingredients on the levels of antioxidant enzymes CAT and SOD regulated downstream of NRF2.

[0099] Figure 3 UV irradiation (10mJ / cm 2 ) and saffron oil-soluble extracts (0.1%, 0.2%, 0.5%) treated for 24 hours on gene expression in HaCat cells. Figure 4 UV irradiation (10mJ / cm 2) and saffron oil-soluble extract (0.5%) for 24 hours to evaluate the expression of CAT, SOD1, and SOD2 in HaCat cells (*VS NC group, #VS UV group). It can be seen that after treatment with saffron oil-soluble extract, all genes were significantly increased compared to the UV group.

Claims

1. A method for preparing an oil-soluble extract of saffron or chicory, comprising the following steps: (1) Freezing and wall breaking: soaking the saffron or chicory raw material in water, followed by multiple freeze-thaw cycles to obtain a first liquid; (2) First medium temperature extraction: add vegetable oil to the first liquid to form a second liquid, and stir and extract. (3) First demulsification: the second liquid is centrifuged to demulsify to separate a first oil phase and a first water phase; the first water phase is mixed with new vegetable oil and subjected to the first medium-temperature extraction; (4) Second medium-temperature extraction: adding the first oil phase to the newly frozen-thawed feed liquid to form a third feed liquid, and stirring and extracting again; (5) Second demulsification: centrifugation is performed again to separate the second oil phase, and a desiccant is added to remove the residual water, and the oil-soluble extract is obtained by filtration; The stirring extraction temperature in steps (2) and (4) is 50-60° C., and the stirring extraction time is 1-2 h; The centrifugal demulsification speed in steps (3) and (5) is 6000-8000 rpm, and the centrifugal demulsification time is 20-30 min.

2. The preparation method according to claim 1, wherein: The saffron or chicory raw material in step (1) is saffron or chicory powder obtained by crushing saffron or chicory and passing it through a 40-60 mesh sieve.

3. The preparation method according to claim 1, wherein: The freeze-thaw process in step (1) is as follows: freezing at -18 to -80°C for 6 to 24 hours and thawing at 50 to 60°C.

4. The preparation method according to claim 1, wherein: The vegetable oil in step (2) is one or more of sunflower oil, peanut oil, corn oil, soybean oil, rapeseed oil and palm oil.

5. The preparation method according to claim 1, wherein: The desiccant in step (5) is one or more of anhydrous calcium sulfate, anhydrous calcium chloride, anhydrous sodium sulfate, and molecular sieve.

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