Extraction method of lycium barbarum seed oil and application of lycium barbarum seed oil in preparation of products for protecting eyes and improving eyesight

Abstract

According to the extraction method of the boxthorn seed oil and the application of the boxthorn seed oil in preparation of the eye-protecting and eyesight-improving product, through compounding of the boxthorn seed oil, the olive oil and the zeaxanthin and innovation of the preparation process, the boxthorn seed oil, the olive oil and the zeaxanthin have a synergistic effect on the eye-protecting and eyesight-improving effects, and meanwhile the bioavailability of the boxthorn seed oil is improved. An extraction system of the eutectic solvent and the enzyme preparation can hydrolyze cell walls of the lycium barbarum seeds, a net structure composed of cellulose is destroyed, and oily components in the lycium barbarum seeds are promoted to be released. Meanwhile, the deep-eutectic solvent has a protective effect on enzyme, so that the enzyme preparation has better hydrolytic activity under proper extraction conditions. According to the method, the oil yield of the wolfberry seed oil and the content of related active ingredients are greatly improved. On the other hand, the olive oil can significantly improve the intake of the active ingredients in the lycium seed oil by retinal cells, and the bioavailability of the lycium seed oil is improved.

Description

Technical Field

[0001] The invention belongs to the field of functional foods, and particularly relates to a method for extracting wolfberry seed oil and application of the wolfberry seed oil in preparing eye-protecting and vision-improving products. Background Art

[0002] With the rapid development of the information age, many visual display terminal devices, including smart phones and tablet computers, are used more and more widely. When people stare at the information on the display device for a long time, the display device continuously emits blue light, and the metabolism of the extraocular muscles and ciliary muscles increases, the burden on the eyes continues to increase, and a series of manifestations mainly based on subjective visual perception will appear, such as blurred vision, dryness, stinging, foreign body sensation in the eyes, frequent blinking, and accompanied by symptoms such as headache and dizziness. The retina is an important tissue organ for the eyeball to receive light energy and form vision. The focus of visible light on the retina produces high oxygen pressure and high concentration, which is prone to lipid peroxidation reactions and its products engulf retinal pigment epithelial cells, causing damage to the retina. In particular, blue light has extremely high energy and can penetrate the lens directly to the retina, causing atrophy or even death of retinal pigment epithelial cells. The macula located in the center of the retina is the most sensitive area for vision, containing cones responsible for vision and color vision. This area contains more pigments such as lutein, while blue light radiation and excessive eye use will produce a large number of free radicals and other peroxidation substances in the retinal cells. Free radicals will consume the pigment components in the macular area. Untimely supply of these nutrients will cause the recovery time of the macula and retina to be prolonged, causing damage to the muscle cell structure and decreased function, leading to sore eyes, blurred vision, etc.

[0003] At present, the extraction methods of wolfberry seed oil are generally pressing, leaching or supercritical fluid extraction, which have the problems of low oil yield, residual organic solvent, high equipment requirements and high cost. At the same time, the active ingredients in the oil are lost greatly. Therefore, the preparation method of wolfberry seed oil is also the focus of the present invention. Summary of the invention

[0004] The invention provides a method for extracting wolfberry seed oil and application of the oil in preparing an eye-protecting and eyesight-improving product. Through the compounding of wolfberry seed oil, olive oil and zeaxanthin and innovation of the preparation process, a synergistic effect is achieved on the eye-protecting and eyesight-improving effects and the bioavailability is improved at the same time.

[0005] The invention firstly provides a method for extracting wolfberry seed oil, wherein the method uses a low eutectic solvent to extract wolfberry seed oil from wolfberry seed powder, and the low eutectic solvent contains choline chloride and glycerol.

[0006] Preferably, the molar ratio of choline chloride to glycerol is 1:2.

[0007] Furthermore, in the method, a citrate buffer containing a hydrolase is also added to the deep eutectic solvent;

[0008] Furthermore, the hydrolase is preferably cellulase.

[0009] As a specific record of the embodiment, the volume ratio of the deep eutectic solvent to the citrate buffer is 1:9;

[0010] In another aspect, the present invention further provides a wolfberry seed oil composition, comprising wolfberry seed oil prepared by the above method and olive oil;

[0011] The mass ratio of the wolfberry seed oil to the olive oil is 1-9:9-1.

[0012] As a specific record of the embodiment, the mass ratio of wolfberry seed oil to olive oil is 1:4.

[0013] Furthermore, the olive oil is completely or partially replaced by other oils.

[0014] As an optional manner, the other oil is at least one of linseed oil, soybean oil, peanut oil, sunflower seed oil, peony seed oil, rapeseed oil, coconut oil, corn oil, and krill oil.

[0015] Furthermore, zeaxanthin is also added to the wolfberry seed oil composition.

[0016] As an optional mode, the added amount of zeaxanthin is 1%.

[0017] Since the oil in wolfberry seeds is mainly present in wolfberry seed cells, and wolfberry seed cells contain a reticular structure composed of cellulose, these reticular structures block the release of oily substances in wolfberry seed cells. In the present invention, the extraction system of the low eutectic solvent and the enzyme preparation can hydrolyze the cell wall of wolfberry seeds, destroy the reticular structure composed of cellulose, and promote the release of oily components in wolfberry seeds. At the same time, the low eutectic solvent has a protective effect on the enzyme, which can make the enzyme preparation have better hydrolysis activity under suitable extraction conditions. This method greatly improves the oil yield of wolfberry seed oil and the content of related active ingredients. On the other hand, olive oil can significantly increase the intake of active ingredients in wolfberry seed oil by retinal cells and improve its bioavailability. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The activity diagram of cellulase in the low eutectic solvent-buffer-cellulase system and the buffer-cellulase system after storage at room temperature for one week.

[0019] Figure 2 This is a graph showing the improvement rate of relieving visual fatigue symptoms of the products in the embodiments of the present invention and the comparative examples.

[0020] Figure 3 Immunohistochemical staining of SERPINF1 and PNPLA2 in mouse retina. DETAILED DESCRIPTION

[0021] The present invention firstly provides a low eutectic solvent for extracting wolfberry seed oil. The solvent system can effectively retain the activity of cellulase, thereby improving the effect of extracting wolfberry seed oil and obtaining a higher content of components with eyesight-improving effect.

[0022] The present invention is described in detail below in conjunction with embodiments and drawings.

[0023] Example 1: Preparation of wolfberry seed oil using a deep eutectic solvent and an enzyme

[0024] This embodiment uses a deep eutectic solvent and enzyme-assisted extraction of wolfberry seed oil, including the following steps:

[0025] (1) Prepare 50 mM citric acid-sodium citrate buffer, pH = 5.0;

[0026] (2) Dissolving cellulase in the buffer prepared in step (1) to obtain a buffer-cellulase system.

[0027] (3) Dry choline chloride solid and glycerol in a molar ratio of 1:2 were mixed in a stoppered conical flask and stirred at 80° C. for 2 h to form a uniform transparent liquid.

[0028] (4) The low eutectic solvent prepared in step (3) is mixed with the buffer prepared in step (1) at a volume ratio of 10% (volume ratio of 1:9) to dissolve the cellulase, thereby obtaining a low eutectic solvent-buffer-cellulase system containing 2% enzyme.

[0029] (5) 40 mL of the low eutectic solvent-buffer-cellulase system prepared in step (4) was placed in a conical flask, 10 g of wolfberry seed powder passed through a 60-mesh sieve was added, heated in a 50° C. water bath, magnetically stirred at a speed of 150 r / min, and extracted for 2 h.

[0030] (6) Add water to the product obtained in step (5) and let it stand, and the resulting oil layer is wolfberry seed oil.

[0031] The low eutectic solvent-buffer-cellulase system and the buffer-cellulase system in this example were stored at room temperature for one week, and the activity of the cellulase therein was detected. Figure 1 As shown, the low eutectic solvent used in this embodiment can significantly protect the hydrolysis activity of cellulase compared with other low eutectic solvents, thereby increasing the oil yield of wolfberry seed oil and the content of related active ingredients.

[0032] Example 2

[0033] The main difference between this embodiment and embodiment 1 is that the enzyme used is Viscozyme@L, which is a composite plant hydrolase produced by Novozymes, including arabinanase, cellulase, b-glucanase, hemicellulase and xylanase.

[0034] Comparative Example 1

[0035] This comparative example mainly studies the effect of the presence of enzymes on the preparation of wolfberry seed oil, and includes the following steps:

[0036] (1) Prepare 50 mM citric acid-sodium citrate buffer, pH = 5.0;

[0037] (2) Dry choline chloride solid and glycerol in a molar ratio of 1:2 were mixed in a stoppered conical flask and stirred at 80° C. for 2 h to form a uniform transparent liquid.

[0038] (3) The low eutectic solvent prepared in step (2) is mixed with the buffer prepared in step (1) at a volume ratio of 10% to obtain a low eutectic solvent-buffer system.

[0039] (4) Place 40 mL of the low eutectic solvent-buffer system prepared in step (3) in a conical flask, add 10 g of wolfberry seed powder passed through a 60-mesh sieve, heat in a 50° C. water bath, stir with a magnetic stirrer at a speed of 150 r / min, and extract for 2 h.

[0040] (5) adding water to the product obtained in step (4) and allowing to stand, and the obtained oil layer is wolfberry seed oil.

[0041] Comparative Example 2

[0042] This comparative example mainly compares the effect of DES on the preparation of wolfberry seed oil, and includes the following steps:

[0043] (1) Prepare 50 mM citric acid-sodium citrate buffer, pH = 5.0;

[0044] (2) Dissolving cellulase in the buffer prepared in step (1) to obtain a buffer-cellulase system containing 2% cellulase.

[0045] (3) Place 40 mL of the buffer solution-cellulase system prepared in step (2) in a conical flask, add 10 g of wolfberry seed powder passed through a 60-mesh sieve, heat in a 50°C water bath, stir with a magnetic stirrer at a speed of 150 r / min, and extract for 2 h.

[0046] (4) adding water to the product obtained in step (3) and allowing to stand, and the obtained oil layer is wolfberry seed oil.

[0047] Comparative Example 3

[0048] This comparative example mainly compares the effects of different DES on the preparation of wolfberry seed oil, including the following steps:

[0049] (1) Prepare 50 mM citric acid-sodium citrate buffer, pH = 5.0;

[0050] (2) Dissolving cellulase in the buffer prepared in step (1) to obtain a buffer-cellulase system.

[0051] (3) Lactic acid and 1,2-propylene glycol in a molar ratio of 1:2 were mixed in a stoppered conical flask and stirred at 80° C. for 2 h to form a uniform transparent liquid.

[0052] (4) The low eutectic solvent prepared in step (3) is mixed with the buffer prepared in step (1) at a volume ratio of 10% to dissolve the cellulase, thereby obtaining a low eutectic solvent-buffer-cellulase system containing 2% enzyme.

[0053] (5) 40 mL of the low eutectic solvent-buffer-cellulase system prepared in step (4) was placed in a conical flask, 10 g of wolfberry seed powder passed through a 60-mesh sieve was added, heated in a 50° C. water bath, magnetically stirred at a speed of 150 r / min, and extracted for 2 h.

[0054] (6) Add water to the product obtained in step (5) and let it stand, and the resulting oil layer is wolfberry seed oil.

[0055] Comparative Example 4

[0056] This comparative example mainly compares the effects of different DES on the preparation of wolfberry seed oil, including the following steps:

[0057] (1) Prepare 50 mM citric acid-sodium citrate buffer, pH = 5.0;

[0058] (2) Dissolving cellulase in the buffer prepared in step (1) to obtain a buffer-cellulase system.

[0059] (3) Dry choline chloride solid and glucose in a molar ratio of 2:1 were mixed in a stoppered conical flask and stirred at 80° C. for 2 h to form a uniform transparent liquid.

[0060] (4) The low eutectic solvent prepared in step (3) is mixed with the buffer prepared in step (1) at a volume ratio of 10% to dissolve the cellulase, thereby obtaining a low eutectic solvent-buffer-cellulase system containing 2% enzyme.

[0061] (5) 40 mL of the low eutectic solvent-buffer-cellulase system prepared in step (4) was placed in a conical flask, 10 g of wolfberry seed powder passed through a 60-mesh sieve was added, heated in a 50° C. water bath, magnetically stirred at a speed of 150 r / min, and extracted for 2 h.

[0062] (6) Add water to the product obtained in step (5) and let it stand, and the resulting oil layer is wolfberry seed oil.

[0063] Example 3

[0064] In this example, the yield of wolfberry seed oil was determined by referring to "GB / T14488.1 Determination of Oil Content of Vegetable Oils" to determine the oil content of wolfberry seeds, and the oil yield was calculated according to the following formula: wolfberry seed oil yield = extracted wolfberry seed oil mass / (wolfberry seed raw material mass × oil content) × 100%

[0065] Example 4

[0066] In this embodiment, the contents of zeaxanthin, β-carotene and lutein in the obtained wolfberry seed oil are determined by reverse phase high performance liquid chromatography, comprising the following steps:

[0067] (1) Accurately weigh 2 g (accurate to 0.1 mg) of the obtained wolfberry seed oil and place it in a 250 mL conical flask, and add 1 g of ascorbic acid, 0.2 g of BHT and 30 mL of anhydrous ethanol.

[0068] (2) Add 30 mL of potassium hydroxide solution to the solution obtained in step (1), shake while adding, cover the bottle with a stopper, place in a constant temperature oscillating box preheated to (54±2)°C, and saponify for 45 minutes. After saponification, immediately cool to room temperature with cold water.

[0069] (3) Prepare the extraction solvent: dissolve 1 g of BHT in 200 mL of cyclohexane, add 400 mL of ether and 400 mL of n-hexane.

[0070] (4) The saponified solution obtained in step (2) was transferred to a 250 mL separatory funnel, 60 mL of the extraction solvent obtained in step (3) was added, the bottle stopper was covered, and the mixture was shaken at room temperature for 10 min and allowed to stand to separate the layers. The aqueous phase was transferred to another separatory funnel and subjected to a second extraction according to this step.

[0071] (5) Combine the organic phases obtained in step (4), wash with 50 mL of water until neutral, and filter and dehydrate through anhydrous sodium sulfate.

[0072] (6) The filtrate obtained in step (5) was concentrated under reduced pressure on a rotary evaporator (40±2)°C to near dryness (not to be evaporated to dryness) and fully dissolved with 0.1% BHT ethanol solution and the volume was adjusted to 10 mL.

[0073] (7) The solution obtained in step (6) was subjected to liquid chromatography. The chromatographic conditions were: the chromatographic column was a C30 chromatographic column, 250 mm × 4.6 mm (inner diameter), particle size 5 μm; injection volume 50 μL; column temperature 30.0°C; flow rate 1.0 mL / min; detection wavelength 445 nm. Mobile phase A was methanol: water (88:12, V / V, containing 0.1% BHT), and mobile phase B was methyl tert-butyl ether (containing 0.1% BHT). Gradient elution was as shown in the following table:

[0074] Table 1: Mobile phase gradient elution program

[0075] Time (min) Mobile phase A Mobile phase B 0 85 15 10 70 30 30 20 80 31 85 15

[0076] The wolfberry seed oil obtained in the above examples and comparative examples was tested, and the test indicators included the yield of wolfberry seed oil and the contents of related active ingredients such as zeaxanthin, β-carotene and lutein. The test results are shown in Table 2.

[0077] Table 2: Yield of wolfberry seed oil extracted by different processes and content of related active ingredients

[0078]

[0079] As shown in Table 2, the oil yield and active component content can be significantly improved by treating the wolfberry seed sample with a low eutectic solvent and an enzyme.

[0080] Example 5

[0081] This embodiment provides a wolfberry seed oil-olive oil composition, wherein a specific preparation process of the product is as follows:

[0082] (1) 1 part of wolfberry seed oil prepared in Example 2 and 4 parts of olive oil were mixed and stirred to prepare a solvent;

[0083] (2) Add zeaxanthin to the solvent in step (1) at a concentration of 1%.

[0084] Example 6

[0085] This example uses a cell experiment to evaluate the effect of olive oil on the bioavailability of zeaxanthin, lutein and β-carotene in wolfberry seed oil. The main implementation steps are as follows:

[0086] Mouse retinal epithelial cells (ARPE-19) were cultured at 3×10 5 / mL concentration was inoculated on a 6-well plate in high-glucose complete medium and cultured for 24 hours. Then the cells were treated with wolfberry seed oil, olive oil, wolfberry seed oil and olive oil for 48 hours.

[0087] The cells were digested and centrifuged together with the culture medium, the supernatant was taken, and the cells were washed twice with cold phosphate buffer. The supernatants were combined, and propanol: dichloromethane (2:1 v / v) was added for 30 min to extract zeaxanthin, lutein and β-carotene in wolfberry seed oil. The extract was dried under nitrogen, and the dried extract was resuspended in 1200 μL of mobile phase. The content of zeaxanthin, lutein and β-carotene therein was detected using the liquid phase conditions of Example 4. The test results are shown in Table 3.

[0088] Table 3: Cellular uptake rate of zeaxanthin, lutein and β-carotene (%)

[0089]

[0090]

[0091] As shown in Table 3, olive oil can significantly increase the uptake of active ingredients in wolfberry seed oil by retinal epithelial cells and improve its bioavailability.

[0092] Example 7

[0093] This example refers to the “Visual Fatigue Relief Test Method” in the “Health Food Function Test and Evaluation Method (2023 Edition)” to evaluate the eyesight-improving effects of the products obtained in Examples 1, 2, 5 and Comparative Examples 1-4.

[0094] The visual acuity improvement rate of each product is as follows: Figure 2 As shown. Compared with the control group, the symptom improvement rate of each embodiment group and the comparative example group was improved, and the difference was significant. The symptom improvement rate of Example 5 was the best, and the difference was extremely significant, indicating that olive oil and wolfberry seed oil have the characteristics of synergistic enhancement, and their effect of relieving visual fatigue is the best.

[0095] Example 8

[0096] This example establishes a visual impairment mouse model and evaluates the eye-improving effect of the wolfberry seed oil-olive oil composition, and the specific implementation is as follows:

[0097] Fifty mice (half male and half female) were randomly selected after adaptive feeding for 7 days, and 5% sodium iodate was intraperitoneally injected at a dose of 25 mg / kg body weight once a day for 6 weeks to construct a visual impairment mouse model of retinal damage; then 10 mice (half male and half female) were selected and intraperitoneally injected with an equal volume of normal saline as the CK group.

[0098] The mice with successful modeling were taken, half male and half female, and randomly divided into a model group and a sample (wolfberry seed oil-olive oil composition prepared in Example 5) group, with normal saline as the CK group. The sample group was gavaged at a dose of 200 mg / kg body weight, once a day; the model group and the CK group were gavaged with the same volume of normal saline, and the gavage was continued for 28 days. During the experiment, the mice were free to eat and drink water. After fasting but not water for 12 hours after the last gavage, the mice were intramuscularly injected with 75 mg / kg body weight of Shutai. After deep anesthesia of the mice, the bilateral eyeballs were removed, fixed with 4% paraformaldehyde solution for 24 hours, and rinsed with running water for 12 hours. After tissue trimming, alcohol dehydration, xylene transparency and paraffin embedding, the slicer was sliced ​​continuously with a thickness of 7 μm, and the slices were dried and stained with HE, and sealed. The sections were then dewaxed with xylene, rehydrated with gradient alcohol, and repaired with citrate buffer. The steps in the immunohistochemistry kit instructions were followed, with incubation in endogenous peroxidase blocker for 20 minutes, incubation in sheep non-immune serum for 20 minutes, incubation in biotin-labeled sheep anti-rabbit IgG for 25 minutes, and incubation in streptavidin-peroxidase for 25 minutes. Rabbit anti-SERPINF1 and PNPLA2 polyclonal antibodies were added after sheep non-immune serum, incubated in a 4°C refrigerator for 24 hours, and DAB was added for color development for 5 minutes. The sections were rinsed once with PBS phosphate buffer for 3 minutes, and then counterstained with hematoxylin after 3 consecutive rinses. After the blank control group of immunohistochemistry staining was incubated with sheep non-immune serum, antibody diluent was added instead of rabbit anti-2 antibodies, and other steps were the same as above. Immunohistochemistry staining was used for image analysis and SERPINF1 and PNPLA2 positive staining localization. The blank control group of immunohistochemistry staining was used to determine the specificity of SERPINF1 and PNPLA2 staining. After the completion of immunohistochemistry staining, the slides were prepared and the results were recorded under a microscope.

[0099] SERPINF1 and PNPLA2 are strongly expressed in mouse ocular tissues, and their loss or dysfunction may impair vision. Figure 3 As shown, after immunohistochemical staining of SERPINF1 and PNPLA2 in the mouse retina, the cell bodies of each layer of the structure were stained light blue, the nuclei were stained dark blue, and the immunopositive products were stained brown, tan or light yellow. The inner plexiform layer and outer plexiform layer of the retina of the model group mice were stained yellow-brown, and the nuclei of the inner nuclear layer were irregular in morphology; the wolfberry seed oil-olive oil composition sample group of the mouse retina prepared in Example 5 had clear boundaries of 9 layers of structure, and the SERPINF1 and PNPLA2 immunopositive products were evenly distributed and light yellow, indicating that the wolfberry seed oil-olive oil composition can effectively protect the damaged retinal nerve fiber layer and photoreceptor cells of mice.

Claims

1. A method for extracting wolfberry seed oil, characterized in that: The method uses a low eutectic solvent to extract wolfberry seed oil from wolfberry seed powder, and the low eutectic solvent contains choline chloride and glycerol.

2. The method according to claim 1, characterized in that The molar ratio of the choline chloride to the glycerol is 1:

2.

3. The method according to claim 1, characterized in that In the method, a citrate buffer containing a hydrolase is also added to the deep eutectic solvent.

4. The method according to claim 3, characterized in that The hydrolase is cellulase.

5. The method according to claim 3, characterized in that The volume ratio of the low eutectic solvent to the citrate buffer is 1:

9.

6. A wolfberry seed oil composition, characterized in that The wolfberry seed oil composition comprises wolfberry seed oil prepared by the method according to claim 1 and olive oil.

7. The wolfberry seed oil composition according to claim 6, wherein The mass ratio of the wolfberry seed oil to the olive oil is 1-9:9-1.

8. The wolfberry seed oil composition according to claim 6, wherein Zeaxanthin is also added into the wolfberry seed oil composition.

9. Use of the wolfberry seed oil composition according to any one of claims 6 to 8 in the preparation of products for protecting eyes and improving vision.

10. A product for protecting eyes and improving vision, characterized in that: The product contains the wolfberry seed oil composition according to any one of claims 6 to 8 in a pharmacologically effective concentration.

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