Preparation method of ginkgo biloba extract as well as product and application of ginkgo biloba extract

Abstract

The invention relates to a preparation method of a ginkgo leaf extract as well as a product and application thereof, the preparation method comprises the following steps: mixing a ginkgo leaf raw material with a compound enzyme for enzymolysis, alcohol extraction and filtration, first separation and purification, fine extraction, second separation and purification, and flocculation and drying to obtain the ginkgo leaf extract, wherein the compound enzyme comprises a combination of beta-glucosidase, xylanase, hemicellulase and pectinase. A specific extraction and separation process is adopted, and a specific enzyme combination is further adopted, so that the four kinds of enzyme combination can interact, the synergistic promotion effect is achieved on improving the enzymolysis extraction effect and improving the moisturizing, anti-inflammatory and anti-oxidation effects of the extract, and the prepared extract has excellent water solubility and can be used for preparing the health-care food. The composition is very suitable for preparing moisturizing and anti-oxidation products, and is mild, non-irritant and good in stability.

Description

Technical Field

[0001] The invention belongs to the field of plant extracts, and specifically relates to a preparation method of a ginkgo leaf extract and a product and application thereof. Background Art

[0002] Ginkgo biloba L. is a dried leaf of Ginkgo biloba L., a plant of the Ginkgoaceae family. The leaves are harvested in autumn when they are still green and dried in time. They are usually cultivated artificially. Ginkgo biloba is a large deciduous tree with long branches and slow-growing short branches. Ginkgo leaves are alternate, scattered radially on long branches, and clustered in 3 to 5 on short branches. They have slender petioles, fan-shaped, light green on both sides, with more or less notches or two cracks on the broad top edge, 5 to 8 (~15) cm wide, and with many forked and spur-shaped veins.

[0003] Ginkgo biloba has high medicinal value, and has the effects of promoting blood circulation and removing blood stasis, dredge meridians and relieve pain, astringe the lungs and relieve asthma, remove turbidity and reduce lipids. It can be used as a medicinal component to relieve or treat blood stasis and obstruction of meridians, chest pain and heart pain, hemiplegia due to stroke, cough and asthma due to lung deficiency, and hyperlipidemia. Ginkgo biloba is rich in flavonoids and terpenoid lactone compounds and other active substances, which have significant effects of dilating blood vessels, protecting vascular endothelial tissue, regulating blood lipids, protecting low-density lipoprotein, inhibiting PAF (platelet activating factor), inhibiting thrombosis, and scavenging free radicals.

[0004] In addition, ginkgo leaf extract also has important application value in cosmetics. Peroxides and free radicals on the body surface are one of the main reasons for accelerating skin browning, promoting skin aging, and forming age spots. To delay the oxidative aging of the skin, peroxides and free radicals on the skin surface must be removed in time. Ginkgo leaf extract has superoxide dismutase (SOD) activity, which is an excellent free radical scavenger and has the effect of promoting blood circulation. Therefore, adding this extract to skin care cosmetics can make the skin moisturized, shiny, and reduce the formation of melanin. Therefore, it is very necessary to provide a new, safe, environmentally friendly, and high-purity preparation method for ginkgo leaf extract. Summary of the invention

[0005] In view of the deficiencies of the prior art, the object of the present invention is to provide a preparation method of Ginkgo biloba extract and its product and application.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a ginkgo leaf extract, the method comprising the following steps:

[0008] S1, crude extraction step: mixing the ginkgo leaf raw material with the composite enzyme for enzymolysis, refluxing the enzymolysis solution with an ethanol aqueous solution, concentrating the reflux solution to remove alcohol, mixing the concentrate with water and heating, standing the mixed solution for filtration, and collecting the filtrate to obtain a crude extract;

[0009] S2, the first separation and purification step: the crude extract is enriched with a macroporous resin to obtain a first eluate;

[0010] S3, a refined extraction step: concentrating and drying the first eluate to obtain a crude extract of ginkgo leaves, performing hot extraction with an ethanol aqueous solution, cooling, filtering, and collecting the filtrate to obtain a refined extract;

[0011] S4, a second separation and purification step: enriching the refined extract with a polyamide resin and a macroporous resin in sequence to obtain a second eluate;

[0012] S5, flocculation step: the second eluate is allowed to stand, filtered, and then the filtrate is collected, and the filtrate is mixed with a flocculant to obtain a flocculation treatment liquid;

[0013] S6, drying step: centrifugally filtering the flocculated solution, collecting the filtrate and drying it to obtain the ginkgo leaf extract;

[0014] Wherein, the complex enzyme comprises a combination of β-glucosidase, xylanase, hemicellulase and pectinase.

[0015] The present invention creatively finds that the composite enzyme composed of four enzymes, namely, beta-glucosidase, xylanase, hemicellulase and pectinase, can interact with each other and positively influence each other, thereby improving the extraction efficiency of the ginkgo leaf raw material and thus increasing the active ingredients in the extract product.

[0016] Furthermore, the preparation method of the ginkgo leaf extract involved in the present invention creatively adopts a specific extraction and separation purification process to extract the ginkgo leaf treatment liquid after enzymatic pretreatment, and combines it with subsequent flocculant treatment, so as to efficiently extract the effective ingredients in the ginkgo leaf, and prepare a high-purity and high-activity ginkgo leaf extract product with extremely strong water solubility, and can be further used in cosmetics to improve the moisturizing, anti-inflammatory and antioxidant effects of the product.

[0017] Preferably, the mass ratio of the β-glucosidase, xylanase, hemicellulase and pectinase is (1-5):(2-4):(2-6):(0.5-3);

[0018] The specific values ​​of "1-5" mentioned above may be, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.;

[0019] The specific values ​​of the above “2-4” may be, for example, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.5, 3.8, 4, etc.;

[0020] The specific values ​​in the above “2-6” may be, for example, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, etc.;

[0021] The specific numerical values ​​in the above "0.5-3" can be, for example, 0.5, 0.8, 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, etc. Other specific point values ​​within this numerical range can be selected, and they will not be elaborated here one by one.

[0022] In the present invention, when the dosage ratio of each enzyme in the composite enzyme combination is within the above specific range, the enzymatic extraction effect on ginkgo leaves is better.

[0023] Preferably, before the enzymatic hydrolysis, the ginkgo leaf raw material is mixed with water, and the solid-to-liquid ratio is 1:(8-15) g / mL, for example, it can be 1:8 g / mL, 1:9 g / mL, 1:10 g / mL, 1:11 g / mL, 1:12 g / mL, 1:13 g / mL, 1:14 g / mL, 1:15 g / mL, etc. Other specific point values ​​within this numerical range can be selected, and they will not be repeated here.

[0024] In the present invention, when the liquid ratio of the ginkgo leaf raw material to water is within the above specific range, a better enzymatic hydrolysis effect is achieved.

[0025] Preferably, the added amount of the complex enzyme is 0.1‰-0.5‰ of the mass of the ginkgo leaf raw material, for example, it can be 0.1‰, 0.15‰, 0.2‰, 0.25‰, 0.3‰, 0.35‰, 0.4‰, 0.45‰, 0.5‰, etc. Other specific point values ​​within this numerical range can be selected, which will not be repeated here.

[0026] In the present invention, when the added amount of the complex enzyme is within the above specific range, the extraction effect on the ginkgo leaf raw material is better.

[0027] Preferably, the temperature of the enzymatic hydrolysis is 40-60°C, for example, it can be 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 52°C, 55°C, 58°C, 59°C, 60°C, etc.; the time is 0.5-2h, for example, it can be 0.5h, 0.8h, 1h, 1.2h, 1.5h, 1.8h, 2h, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described one by one here.

[0028] Preferably, the liquid ratio of the ginkgo leaf raw material to the ethanol aqueous solution in step S1 is 1:(4-10) g / mL, for example, it can be 1:4 g / mL, 1:5 g / mL, 1:6 g / mL, 1:7 g / mL, 1:8 g / mL, 1:9 g / mL, 1:10 g / mL, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described one by one here.

[0029] Preferably, the volume percentage of ethanol in the ethanol aqueous solution in step S1 is 30%-90%, for example, it can be 30%, 40%, 50%, 60%, 70%, 80%, 90%, etc. Other specific point values ​​within this numerical range can be selected and will not be described one by one here.

[0030] Preferably, the number of reflux extractions in step S1 is 2-5 times, and the time of reflux extraction is 1-3h / time, for example, it can be 1h / time, 1.2h / time, 1.5h / time, 2h / time, 2.5h / time, 3h / time, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described one by one here.

[0031] Preferably, the volume ratio of the concentrate to water in step S1 is 1:(2-20), for example, it can be 1:2, 1:4, 1:6, 1:8, 1:10, 1:12, 1:14, 1:16, 1:18, 1:20, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0032] Preferably, the temperature of the mixing and heating with water in step S1 is 50-100°C, for example, it can be 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 98°C, 100°C, etc.; the time is 20-60min, for example, it can be 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, 60min, etc., and other specific point values ​​within the numerical range can be selected, which will not be described here one by one.

[0033] Preferably, the standing temperature in step S1 is 2-6°C, for example, 2°C, 2.5°C, 3°C, 3.5°C, 4°C, 4.5°C, 5°C, 5.5°C, 6°C, etc.; the time is 18-48h, for example, it can be 18h, 19h, 20h, 21h, 22h, 24h, 25h, 26h, 28h, 30h, 32h, 35h, 38h, 40h, 45h, 48h, etc. Other specific point values ​​within the numerical range can be selected, and they will not be described one by one here.

[0034] Preferably, the type of the macroporous resin in step S2 includes any one of AB-8, D101 or HPD100.

[0035] Preferably, the enrichment in step S2 specifically comprises: loading the crude extract onto a macroporous resin for adsorption, eluting with water, analyzing with a 40%-90% ethanol aqueous solution, and collecting the eluate to obtain.

[0036] Preferably, the elution flow rate is 120-180 L / h, for example, it can be 120 L / h, 130 L / h, 140 L / h, 150 L / h, 160 L / h, 170 L / h, 180 L / h, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0037] Preferably, the temperature of the concentrated drying in step S3 is 60-80°C, for example, it can be 60°C, 65°C, 68°C, 70°C, 72°C, 75°C, 78°C, 80°C, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described one by one here.

[0038] Preferably, the solid-liquid ratio of the crude ginkgo leaf extract to the ethanol aqueous solution in step S3 is 1:(4-10) g / mL, for example, it can be 1:4 g / mL, 1:5 g / mL, 1:6 g / mL, 1:7 g / mL, 1:8 g / mL, 1:9 g / mL, 1:10 g / mL, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described one by one here.

[0039] Preferably, the volume percentage of ethanol in the ethanol aqueous solution in step S3 is 40%-90%, for example, it can be 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, etc. Other specific point values ​​within this numerical range can be selected, which will not be described here one by one.

[0040] Preferably, the temperature of the hot dipping in step S3 is 40-100°C, for example, it can be 40°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 98°C, 100°C, etc.; the time is 20-60min, for example, it can be 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, 60min, etc., and other specific point values ​​within the numerical range can be selected, which will not be described one by one here.

[0041] Preferably, the cooling temperature in step S3 is 2-6°C, for example, 2°C, 2.5°C, 3°C, 3.5°C, 4°C, 4.5°C, 5°C, 5.5°C, 6°C, etc.; the time is 18-48h, for example, it can be 18h, 19h, 20h, 21h, 22h, 24h, 25h, 26h, 28h, 30h, 32h, 35h, 38h, 40h, 45h, 48h, etc. Other specific point values ​​within the numerical range can be selected, and they will not be described one by one here.

[0042] Preferably, the type of the macroporous resin in step S4 includes DM130.

[0043] Preferably, the enrichment specifically comprises: loading the refined extract onto a polyamide resin and a macroporous resin in sequence for adsorption, washing with water to remove impurities, analyzing with a 40%-90% ethanol aqueous solution, and collecting the eluate to obtain.

[0044] Preferably, the refined extract is also concentrated to remove alcohol, the concentrate is mixed with water, cooled and filtered to collect the filtrate before loading.

[0045] Preferably, the volume ratio of the concentrate to water is 1:(10-40), for example, it can be 1:10, 1:12, 1:15, 1:18, 1:20, 1:25, 1:30, 1:35, 1:40, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0046] Preferably, the cooling temperature is 2-6°C, for example, 2°C, 2.5°C, 3°C, 3.5°C, 4°C, 4.5°C, 5°C, 5.5°C, 6°C, etc.; the time is 18-48h, for example, it can be 18h, 19h, 20h, 21h, 22h, 24h, 25h, 26h, 28h, 30h, 32h, 35h, 38h, 40h, 45h, 48h, etc.

[0047] Preferably, the elution flow rate of the polyamide resin is 80-150 L / h, for example, it can be 80 L / h, 85 L / h, 90 L / h, 95 L / h, 100 L / h, 110 L / h, 120 L / h, 125 L / h, 130 L / h, 135 L / h, 140 L / h, 145 L / h, 150 L / h, etc. Other specific point values ​​within the numerical range can be selected, and they will not be described one by one here.

[0048] Preferably, the adsorption time of the macroporous resin is 20-60 min, for example, it can be 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, etc.; the flow rate is 60-100 L / h, for example, it can be 60 L / h, 65 L / h, 70 L / h, 75 L / h, 80 L / h, 85 L / h, 90 L / h, 95 L / h, 100 L / h, etc. Other specific point values ​​within the numerical range can be selected, and they will not be described one by one here.

[0049] Preferably, in step S5, the second eluent is also concentrated and alcohol-removed before being allowed to stand.

[0050] Preferably, the temperature of the cooling treatment in step S5 is 2-6°C, for example, 2°C, 2.5°C, 3°C, 3.5°C, 4°C, 4.5°C, 5°C, 5.5°C, 6°C, etc.; the time is 18-48h, for example, it can be 18h, 19h, 20h, 21h, 22h, 24h, 25h, 26h, 28h, 30h, 32h, 35h, 38h, 40h, 45h, 48h, etc. Other specific point values ​​within the numerical range can be selected, and they will not be described one by one here.

[0051] Preferably, the flocculant in step S5 comprises a combination of gelatin, chitosan and guar gum;

[0052] In the present invention, it is unexpectedly found that when gelatin, chitosan and guar gum are used as flocculants at the same time, inactive impurities in the extract can be removed, and the ginkgo leaf extract obtained after treatment has higher purity and better effect, and has further better effects in anti-oxidation and moisturizing.

[0053] The flocculant is pretreated as follows before use: chitosan and guar gum are first mixed and stirred in a small amount of acetic acid aqueous solution to a paste state, and then mixed with gelatin, water is added, and heated to swell.

[0054] Preferably, the mass ratio of gelatin, chitosan and guar gum is (0.5-2):1:(1-3);

[0055] The above “0.5-2” may be, for example, 0.5, 0.6, 0.8, 1, 1.2, 1.4, 1.5, 1.6, 1.8, 2, etc.;

[0056] The above-mentioned "1-3" can be, for example, 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, etc. Other specific point values ​​within this numerical range can be selected, and they will not be elaborated here one by one.

[0057] Preferably, the amount of flocculant used in step S5 is 0.05‰-0.1‰ of the weight of the filtrate, for example, it can be 0.05‰, 0.055‰, 0.06‰, 0.065‰, 0.07‰, 0.075‰, 0.08‰, 0.085‰, 0.09‰, 0.095‰, 0.1‰, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described here one by one.

[0058] Preferably, the mixing temperature in step S5 is 50-60°C, for example, it can be 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, etc.; the time is 10-30min, for example, it can be 10min, 12min, 14min, 15min, 16min, 18min, 20min, 25min, 28min, 30min, etc., and other specific point values ​​within the numerical range can be selected, which will not be described here one by one.

[0059] Preferably, the centrifugal speed of the flocculation treatment liquid is 2000-4000rpm, for example, it can be 2000rpm, 2250rpm, 2500rpm, 2750rpm, 3000rpm, 3250rpm, 3500rpm, 3750rpm, 4000rpm, etc.; the time is 15-40min, for example, it can be 15min, 18min, 20min, 22min, 25min, 28min, 30min, 35min, 40min, etc., and other specific point values ​​within the numerical range can be selected, which will not be described here one by one.

[0060] In a second aspect, the present invention provides a ginkgo leaf extract, which is prepared by the preparation method described in the first aspect.

[0061] In a third aspect, the present invention provides a use of the ginkgo leaf extract according to the second aspect in the preparation of a moisturizing and / or antioxidant product.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] The present invention develops a new preparation method of ginkgo leaf extract, creatively adopts a specific extraction and separation purification process to extract the ginkgo leaf treatment liquid after enzymatic hydrolysis pretreatment, combines flocculation treatment to remove impurities, and further adopts a composite enzyme combination including β-glucosidase, xylanase, hemicellulase and pectinase for enzymatic hydrolysis. The four enzymes are used in combination, which can interact and positively affect each other, thereby improving the extraction efficiency of the ginkgo leaf raw material, thereby increasing the active ingredients in the extract product. The prepared high-purity and high-activity ginkgo leaf extract product has extremely strong water solubility, which is helpful for further application in cosmetics to improve the moisturizing, anti-inflammatory and antioxidant effects of the product. DETAILED DESCRIPTION

[0064] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0065] In the following examples, unless otherwise specified, all reagents and consumables were purchased from conventional reagent manufacturers in the field; unless otherwise specified, the experimental methods and technical means used were conventional methods and means in the field.

[0066] The product information of β-glucosidase, xylanase, hemicellulase and pectinase involved in the following examples are G824159 (Macklin), X875254 (Macklin), H8112 (Solarbio) and S10008 (Source Leaf), respectively; gelatin, chitosan and guar gum were purchased from Shanghai Aladdin Reagent.

[0067] Example 1

[0068] This embodiment provides a ginkgo leaf extract, and the preparation method thereof is as follows:

[0069] (1) Crude extraction step: Ginkgo biloba leaves are ground and then mixed with pure water at a solid-liquid ratio of 1:12 g / mL, and then mixed with a composite enzyme (composed of β-glucosidase, xylanase, hemicellulase and pectinase in a mass ratio of 3:2:5:1), and enzymolysis is performed at 52°C for 1 hour, wherein the amount of the composite enzyme added is 0.3‰ of the crushed ginkgo leaves. After the reaction is completed, an enzymatic solution is obtained, and a 60% ethanol aqueous solution is added to the enzymatic solution for reflux extraction. The solid-liquid ratio of the ginkgo leaf raw material to the ethanol aqueous solution is 1:8 g / mL. Reflux extraction is performed three times, and the extraction time for each time is 2 hours, 2 hours and 1.5 hours, respectively. The reflux solutions are combined, and the alcohol is removed by reduced pressure concentration at 70°C. The concentrate is mixed with 10 times the volume of pure water, mixed and heated at 80°C for 30 minutes, cooled at 4°C and allowed to stand for 24 hours, and then filtered. The filtrate is collected to obtain a crude extract.

[0070] (2) First separation and purification step: The crude extract was loaded onto a D101 macroporous resin column for enrichment, eluted with pure water at a flow rate of 100 L / h, and analyzed with 75% ethanol aqueous solution to obtain a first eluate.

[0071] (3) Extraction step: The first eluate was concentrated under reduced pressure at 70°C to remove alcohol, and the concentrate was concentrated under reduced pressure at 60°C and dried to obtain a crude extract of ginkgo leaves. The crude extract of ginkgo leaves was hot-extracted with a 75% ethanol aqueous solution at 80°C for 30 min with a solid-liquid ratio of 1:8 g / mL. The mixture was cooled at 4°C for 24 h and then filtered to collect the filtrate to obtain a refined extract.

[0072] (4) Second separation and purification step: The extract was concentrated under reduced pressure at 70°C to remove alcohol, the concentrate was mixed with 25 times the volume of pure water, cooled at 4°C for 24 hours, and then filtered. The filtrate was loaded onto a resin column connected in series with a polyamide resin column (100 L / h) and a DM130 macroporous resin column (80 L / h), adsorbed for 30 minutes, eluted with pure water, and analyzed with 75% ethanol aqueous solution to obtain a second eluate.

[0073] (5) Flocculation step: The second eluate was concentrated under reduced pressure at 70°C to remove alcohol, cooled at 4°C and allowed to stand for 24 hours, then filtered to collect the filtrate; the filtrate was mixed with a flocculant (chitosan and guar gum were mixed with 3 times the mass of 1% acetic acid aqueous solution and stirred until a paste was formed, then mixed with gelatin, and 70 times the mass of water was added to 50°C and mixed and swollen for 18 hours; the mass ratio of gelatin, chitosan and guar gum was 1:1:1) for flocculation treatment, the amount of flocculant used was 0.08‰ of the weight of the filtrate, the flocculation treatment temperature was 55°C, and the time was 15 minutes to obtain a flocculated solution.

[0074] (6) Drying step: the flocculated solution was centrifuged at 2500 rpm for 35 min, the supernatant was filtered using a filter plate with a pore size of 1 μm, and the filtrate was collected and dried to obtain the ginkgo leaf extract.

[0075] Example 2

[0076] This embodiment provides a ginkgo leaf extract, and the preparation method thereof is as follows:

[0077] (1) Crude extraction step: Ginkgo biloba leaves are ground and then mixed with pure water at a solid-liquid ratio of 1:8 g / mL, and then mixed with a composite enzyme (composed of β-glucosidase, xylanase, hemicellulase and pectinase in a mass ratio of 1:3:4:1.5), and enzymolysis is performed at 45°C for 1.6 h, wherein the amount of the composite enzyme added is 0.15‰ of the crushed ginkgo leaves. After the reaction is completed, an enzymatic solution is obtained, and a 40% ethanol aqueous solution is added to the enzymatic solution for reflux extraction. The solid-liquid ratio of the ginkgo leaf raw material to the ethanol aqueous solution is 1:5 g / mL. Reflux extraction is performed twice, and the extraction time for each time is 3 h. The reflux solutions are combined, and the alcohol is removed by reduced pressure concentration at 70°C. The concentrate is mixed with 5 times the volume of pure water, mixed and heated at 60°C for 50 min, cooled at 6°C and allowed to stand for 48 h, and then filtered. The filtrate is collected to obtain a crude extract.

[0078] (2) First separation and purification step: The crude extract was loaded onto a HPD100 macroporous resin column for enrichment, eluted with pure water at a flow rate of 170 L / h, and analyzed with 50% ethanol aqueous solution to obtain a first eluate.

[0079] (3) Extraction step: The first eluate was concentrated under reduced pressure at 70°C to remove alcohol, and the concentrate was concentrated under reduced pressure at 70°C and dried to obtain a crude extract of ginkgo leaves. The crude extract of ginkgo leaves was hot-extracted with a 50% ethanol aqueous solution at 50°C for 50 min, with a solid-liquid ratio of 1:5 g / mL. The mixture was cooled at 6°C for 48 h and then filtered, and the filtrate was collected to obtain a refined extract.

[0080] (4) Second separation and purification step: The extract was concentrated under reduced pressure at 70°C to remove alcohol, the concentrate was mixed with 10 times the volume of pure water, cooled at 6°C for 48 hours and filtered, the filtrate was loaded onto a resin column connected in series with a polyamide resin column (150 L / h) and a DM130 macroporous resin column (100 L / h), adsorbed for 50 minutes, eluted with pure water, and analyzed with 50% ethanol aqueous solution to obtain a second eluate.

[0081] (5) Flocculation step: The second eluate was concentrated under reduced pressure at 70°C to remove alcohol, cooled to 6°C and allowed to stand for 48 hours, then filtered to collect the filtrate; the filtrate was mixed with a flocculant (chitosan and guar gum were mixed with 3 times the mass of 1% acetic acid aqueous solution and stirred until a paste was formed, then mixed with gelatin, and 70 times the mass of water was added to 50°C and mixed and swollen for 18 hours; the mass ratio of gelatin, chitosan and guar gum was 0.5:1:3) for flocculation treatment, the amount of flocculant used was 0.05‰ of the weight of the filtrate, the flocculation treatment temperature was 50°C, and the time was 30 minutes to obtain a flocculated solution.

[0082] (6) Drying step: the flocculated solution was centrifuged at 2000 rpm for 40 min, the supernatant was filtered using a filter plate with a pore size of 1 μm, and the filtrate was collected and dried to obtain the ginkgo leaf extract.

[0083] Example 3

[0084] This embodiment provides a ginkgo leaf extract, and the preparation method thereof is as follows:

[0085] (1) Crude extraction step: Ginkgo biloba leaves are ground and then mixed with pure water at a solid-liquid ratio of 1:14 g / mL, and then mixed with a composite enzyme (composed of β-glucosidase, xylanase, hemicellulase and pectinase in a mass ratio of 5:4:2:3), and enzymolysis is performed at 58°C for 0.6 h, wherein the amount of the composite enzyme added is 0.45‰ of the crushed ginkgo leaves. After the reaction is completed, an enzymolysis solution is obtained, and an 85% ethanol aqueous solution is added to the enzymolysis solution for reflux extraction. The solid-liquid ratio of the ginkgo leaf raw material to the ethanol aqueous solution is 1:10 g / mL. Reflux extraction is performed 4 times, and the extraction time for each time is 1 h, 2 h, 2 h and 1 h, respectively. The reflux solutions are combined, and the alcohol is removed by reduced pressure concentration at 70°C. The concentrate is mixed with 18 times the volume of pure water, mixed and heated at 100°C for 20 min, cooled at 2°C and allowed to stand for 18 h, and then filtered. The filtrate is collected to obtain a crude extract.

[0086] (2) First separation and purification step: The crude extract was loaded onto an AB-8 macroporous resin column for enrichment, eluted with pure water at a flow rate of 120 L / h, and analyzed with 85% ethanol aqueous solution to obtain a first eluate.

[0087] (3) Extraction step: The first eluate was concentrated under reduced pressure at 70°C to remove alcohol, and the concentrate was concentrated under reduced pressure at 80°C and dried to obtain a crude extract of ginkgo leaves. The crude extract of ginkgo leaves was mixed with a 90% ethanol aqueous solution at 90°C for 25 min at a solid-liquid ratio of 1:10 g / mL. The mixture was cooled at 2°C for 30 h and then filtered to collect the filtrate to obtain a refined extract.

[0088] (4) Second separation and purification step: The filtrate of the extract was concentrated under reduced pressure at 70°C to remove alcohol, the concentrate was mixed with 40 times the volume of pure water, cooled at 2°C for 18 hours, and then filtered. The filtrate was loaded onto a resin column connected in series with a polyamide resin column (80 L / h) and a DM130 macroporous resin column (60 L / h), adsorbed for 20 minutes, eluted with pure water, and analyzed with 90% ethanol aqueous solution to obtain a second eluate.

[0089] (5) Flocculation step: The second eluate was concentrated under reduced pressure at 70°C to remove alcohol, cooled at 2°C and allowed to stand for 18 h before filtering to collect the filtrate; the filtrate was mixed with a flocculant (chitosan and guar gum were mixed with 3 times the mass of 1% acetic acid aqueous solution and stirred until a paste was formed, then mixed with gelatin, and 70 times the mass of water was added to 50°C and mixed and swollen for 18 h; the mass ratio of gelatin, chitosan and guar gum was 2:1:2) for flocculation treatment, the amount of flocculant used was 0.1‰ of the weight of the filtrate, the flocculation treatment temperature was 60°C, and the time was 10 min to obtain a flocculated solution.

[0090] (6) Drying step: the flocculated solution was centrifuged at 3500 rpm for 20 min, the supernatant was filtered using a filter plate with a pore size of 1 μm, and the filtrate was collected and dried to obtain the ginkgo leaf extract.

[0091] Example 4

[0092] This embodiment provides a ginkgo leaf extract, which uses the crushed ginkgo leaf of embodiment 1. The only difference between its preparation method and that of embodiment 1 is that the amount of compound enzyme added is 1‰, and the remaining steps and process parameters are all referenced to embodiment 1.

[0093] Example 5

[0094] This embodiment provides a ginkgo leaf extract, which uses the crushed ginkgo leaf of embodiment 1. The only difference between its preparation method and that of embodiment 1 is that the amount of compound enzyme added is 0.05‰, and the remaining steps and process parameters are all referred to embodiment 1.

[0095] Example 6

[0096] This embodiment provides a ginkgo leaf extract, using the second eluent of embodiment 1. The preparation method thereof is different from that of embodiment 1 only in that the flocculant does not contain gelatin, and the reduced amount thereof is proportionally distributed to the chitosan and guar gum components. The remaining steps and process parameters are all referred to embodiment 1.

[0097] Example 7

[0098] This embodiment provides a ginkgo leaf extract, using the second eluent of embodiment 1. The preparation method thereof is different from that of embodiment 1 only in that the flocculant does not contain chitosan, and the reduced amount thereof is proportionally distributed to the gelatin and guar gum components. The remaining steps and process parameters are all referred to embodiment 1.

[0099] Example 8

[0100] This embodiment provides a ginkgo leaf extract, using the second eluent of embodiment 1. The preparation method thereof is different from that of embodiment 1 only in that guar gum is not contained in the flocculant, and the reduced amount thereof is proportionally distributed to the chitosan and gelatin components. The remaining steps and process parameters are all referred to embodiment 1.

[0101] Comparative Example 1

[0102] This comparative example provides a ginkgo leaf extract, which uses the ginkgo leaf crushed material of Example 1. The only difference between its preparation method and that of Example 1 is that the complex enzyme does not contain β-glucosidase, and its reduced portion is proportionally distributed to xylanase, hemicellulase and pectinase. The remaining steps and process parameters are all referred to Example 1.

[0103] Comparative Example 2

[0104] This comparative example provides a ginkgo leaf extract, which uses the ginkgo leaf crushed material of Example 1. The only difference between its preparation method and that of Example 1 is that the complex enzyme does not contain xylanase, and its reduced portion is proportionally distributed to β-glucosidase, hemicellulase and pectinase. The remaining steps and process parameters are all referred to Example 1.

[0105] Comparative Example 3

[0106] This comparative example provides a ginkgo leaf extract, which uses the ginkgo leaf crushed material of Example 1. The only difference between its preparation method and that of Example 1 is that the complex enzyme does not contain hemicellulase, and the reduced amount of hemicellulase is proportionally distributed to β-glucosidase, xylanase and pectinase. The remaining steps and process parameters are all referred to Example 1.

[0107] Comparative Example 4

[0108] This comparative example provides a ginkgo leaf extract, which uses the ginkgo leaf crushed material of Example 1. The only difference between its preparation method and that of Example 1 is that the complex enzyme does not contain pectinase, and its reduced portion is proportionally distributed to β-glucosidase, xylanase and hemicellulase. The remaining steps and process parameters are all referred to Example 1.

[0109] Comparative Example 5

[0110] This comparative example provides a ginkgo leaf extract, which uses the ginkgo leaf crushed material of Example 1. The only difference between its preparation method and that of Example 1 is that no enzymatic hydrolysis treatment is performed. After the ginkgo leaf crushed material is mixed with pure water, it is directly mixed with an ethanol aqueous solution for reflux extraction treatment. The remaining subsequent steps and process parameters are all referred to Example 1.

[0111] Comparative Example 6

[0112] This comparative example provides a ginkgo leaf extract, using the first eluent of Example 1. The difference between the preparation method and Example 1 is that the fine extraction and the second separation and purification treatment are not performed, and the first eluent is used to directly perform the subsequent flocculation and drying steps. The relevant steps and process parameters are all referred to Example 1.

[0113] Comparative Example 7

[0114] This comparative example provides a ginkgo leaf extract, which uses the second eluent of Example 1. The preparation method thereof is different from that of Example 1 only in that no flocculation treatment is performed, the second eluent is concentrated under reduced pressure at 70° C. to remove alcohol, cooled at 4° C. for 24 hours, filtered, the filtrate is collected, and dried to obtain the ginkgo leaf extract.

[0115] Test Example 1

[0116] Antioxidant effect test

[0117] (1) DPPH-free radical scavenging

[0118] A 96-well plate was used, with three replicates per group, and the system was 200 μL. Sample group: 100 μg of the extract samples prepared in Examples 1-8 and Comparative Examples 1-7 were taken, respectively, mixed with 100 μL of pure water, and 100 μL of DPPH solution (0.1 mM) was added to the reaction system; control group: 100 μL of pure water was taken, and then 100 μL of DPPH solution (0.1 mM) was added. After the reaction system was constructed, it was shaken in the dark for 10 minutes, and the absorbance at 517 nm was tested using an ELISA instrument.

[0119] The calculation method of clearance rate is as follows: Clearance rate (%) = [(A0-Ax) / A0] × 100%, A0 is the absorbance of the control group, and Ax is the absorbance of the sample group.

[0120] (2) ABTS-free radical scavenging

[0121] Preparation of ABTS working solution: Mix ABTS and potassium persulfate solution to prepare 7 mM ABTS stock solution, store in dark for 12 h, shake well and mix with 10 mM PBS (pH=7.4) so ​​that the absorbance of the solution at 734 nm is 0.8, to obtain ABTS working solution.

[0122] A 96-well plate was used, with three replicates in each group, and the system was 200 μL. Sample group: 100 μg of the extract samples prepared in Examples 1-8 and Comparative Examples 1-7 were taken, respectively, mixed with 10 μL of pure water, and 190 μL of ABTS working solution was added to the reaction system; control group: 10 μL of pure water was taken, and then 190 μL of ABTS working solution was added. After the reaction system was constructed, it was fully shaken and incubated for 6 minutes, and the absorbance at 734 nm was tested with an enzyme marker.

[0123] The calculation method of clearance rate is as follows: Clearance rate (%) = [(A0-Ax) / A0] × 100%, A0 is the absorbance of the control group, and Ax is the absorbance of the sample group.

[0124] The test results are shown in Table 1.

[0125] Table 1

[0126] Group DPPH-free radical scavenging rate (%) ABTS-free radical scavenging rate (%) Example 1 98.42 95.84 Example 2 96.88 93.69 Example 3 97.13 94.76 Example 4 93.06 90.75 Example 5 92.51 88.79 Example 6 91.85 89.64 Example 7 92.07 90.53 Example 8 89.76 88.34 Comparative Example 1 85.64 83.59 Comparative Example 2 86.66 84.37 Comparative Example 3 85.98 83.91 Comparative Example 4 86.04 84.00 Comparative Example 5 81.59 80.25 Comparative Example 6 74.32 71.38 Comparative Example 7 79.63 76.55

[0127] From the data in the table, we can see that

[0128] (1) It can be seen from Examples 1 to 3 that the ginkgo leaf extract prepared by the present invention has excellent scavenging effects on DPPH-free radicals and ABTS-free radicals, and exhibits significant antioxidant effects, among which Example 1 has the best effect.

[0129] (2) By comparing Example 1 with Examples 4-5, it can be seen that when the amount of the complex enzyme added is within a specific range, the antioxidant effect of the ginkgo leaf extract prepared therefrom is better.

[0130] (3) By comparing Example 1 with Examples 6-8, it can be seen that when the flocculant composed of gelatin, chitosan and guar gum is used to treat the extract in the present invention, the inactive substances and other impurities in the extract can be further removed to prepare a high-purity active ginkgo leaf extract.

[0131] (4) By comparing Example 1 with Comparative Examples 1-4, it can be seen that the present invention has an excellent enzymatic hydrolysis and extraction effect on Ginkgo biloba leaves by using a compound of four enzymes, namely, β-glucosidase, xylanase, hemicellulase and pectinase. When Comparative Examples 1-4 do not contain any of the four enzymes, namely, β-glucosidase, xylanase, hemicellulase and pectinase, the antioxidant effect of the Ginkgo biloba extracts prepared therefrom is lower than that of Example 1, indicating that the four enzymes, namely, β-glucosidase, xylanase, hemicellulase and pectinase, have a synergistic effect when used to extract the active ingredients in Ginkgo biloba leaves.

[0132] (5) By comparing Example 1 with Comparative Examples 5-7, it can be seen that the present invention adopts a specific extraction and impurity removal method of "enzymatic pretreatment-rough extraction-fine extraction-flocculation and impurity removal" to prepare a ginkgo leaf extract product with high purity and high antioxidant activity.

[0133] Test Example 2

[0134] Anti-inflammatory effect test

[0135] Test method: RAW264.7 mouse macrophages in logarithmic growth phase with good morphology were selected and inoculated in DMEM culture medium containing 10% FBS fetal bovine serum and incubated for 24 hours. 5 The cells were seeded at a density of 100 cells / well in a 24-well plate and incubated at 37°C with 5% CO 2 The cells were incubated in an incubator for 2 h and stimulated with 1 μg / mL lipopolysaccharide (LPS) for 24 h to establish a cell inflammation model.

[0136] 0.5% of the ginkgo leaf extract prepared in Examples 1-8 and Comparative Examples 1-7 was added to each group of cells. After incubation at 37°C for 24 hours, the cell supernatant was collected and tested according to the instructions of the ELISA kit and the nitric oxide detection kit, and the contents of TNF-a, NO and IL-6 in the collected cell supernatant were detected respectively. The experimental results were analyzed by data processing software, and the results are shown in Table 2.

[0137] Table 2

[0138]

[0139]

[0140] It can be seen from the data in the table that the ginkgo leaf extract prepared by the preparation method provided by the present invention has excellent anti-inflammatory effect and can significantly inhibit the secretion of related pro-inflammatory factors. The anti-inflammatory effect of Example 1 is better than that of Examples 4-8 and Comparative Examples 1-7, indicating that the specific preparation conditions of the ginkgo leaf extract adopted by the present invention have different degrees of influence on the anti-inflammatory effect of the final extraction product.

[0141] Test Example 3

[0142] Moisturizing effect test

[0143] 120 subjects were selected, with an age range of 28-40 years old and an average age of 34 years old. They were randomly divided into 15 groups. The subjects uniformly cleaned the curved side of the left and right forearms, and after gently blotting with a paper towel, each group of samples was marked with 6 areas of 3cm×3cm as the test area, with at least 2cm between each area, and the test results were statistically averaged. The test area was smeared with an appropriate amount of the diluted samples of Example 1-8 and Comparative Example 1-7 (Ginkgo biloba extract was mixed with pure water, and the final concentration of the extract in the diluted sample was 0.5%). Before using the sample and after using it for 2 hours, the subject data was arranged for collection, and skin parameters were collected each time using a skin test instrument (Corneometer CM825), and the skin stratum corneum moisture content growth rate (%) was statistically calculated, and the growth rate = (moisture content after using it for 2 hours-moisture content before use) / moisture content before use, and the results are shown in Table 3.

[0144] Table 3

[0145] Group Moisture content growth rate (%) Example 1 58.1 Example 2 56.6 Example 3 57.0 Example 4 54.9 Example 5 52.1 Example 6 50.8 Example 7 51.9 Example 8 51.5 Comparative Example 1 48.2 Comparative Example 2 49.3 Comparative Example 3 48.1 Comparative Example 4 47.8 Comparative Example 5 42.6 Comparative Example 6 38.3 Comparative Example 7 41.7

[0146] It can be seen from the data in the table that the ginkgo leaf extract prepared by the preparation method provided by the present invention has excellent moisturizing effect and can maintain the moisture content of the stratum corneum of the skin. The moisturizing effect of Example 1 is better than that of Examples 4-8 and Comparative Examples 1-7, indicating that the specific preparation conditions of the ginkgo leaf extract adopted by the present invention have different degrees of influence on the moisturizing effect of the final extraction product.

[0147] Test Example 4

[0148] Product safety testing

[0149] According to the subject inclusion criteria, 40 females and 40 males were selected to participate in the test, and randomly divided into 8 groups (5 males and 5 females in each group) to receive the trial. Take an appropriate amount of the extract sample prepared in Examples 1-8 (the extract is mixed with pure water to adjust the extract concentration to 4%, and then tested) and place it in the drug chamber of the spot test tape. The spot tester with the sample is applied to the skin of the curved side of the forearm of the subject with medical tape for 24 hours. 30 minutes after removing the test spot tester, observe the skin reaction after the indentation disappears. For those with negative results, observe again 24 hours and 48 hours after the test, and record the skin reaction results. The reaction degree grading standard is as shown in the following table.

[0150]

[0151]

[0152] The results show that the products prepared in Examples 1-8 showed negative reactions 24h and 48h after the test, and there was no irritation or erythema. It can be seen that the ginkgo extract product prepared by the present invention has no irritation to the skin and no positive reaction, which proves that the product is highly safe, less irritating and mild.

[0153] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0154] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0155] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing a ginkgo leaf extract, characterized in that: The preparation method comprises the following steps: S1, crude extraction step: mixing the ginkgo leaf raw material with the composite enzyme for enzymolysis, refluxing the enzymolysis solution with an ethanol aqueous solution, concentrating the reflux solution to remove alcohol, mixing the concentrate with water, heating, standing, filtering the mixed solution, and collecting the filtrate to obtain a crude extract; S2, the first separation and purification step: the crude extract is enriched with a macroporous resin to obtain a first eluate; S3, a refined extraction step: concentrating and drying the first eluate to obtain a crude extract of ginkgo leaves, performing hot extraction with an ethanol aqueous solution, cooling, filtering, and collecting the filtrate to obtain a refined extract; S4, a second separation and purification step: enriching the refined extract with a polyamide resin and a macroporous resin in sequence to obtain a second eluate; S5, flocculation step: the second eluate is allowed to stand, filtered, and then the filtrate is collected, and the filtrate is mixed with a flocculant to obtain a flocculation treatment liquid; S6, drying step: centrifugally filtering the flocculated solution, collecting the filtrate and drying it to obtain the ginkgo leaf extract; Wherein, the complex enzyme comprises a combination of β-glucosidase, xylanase, hemicellulase and pectinase.

2. The preparation method according to claim 1, characterized in that: The mass ratio of the β-glucosidase, xylanase, hemicellulase and pectinase is (1-5):(2-4):(2-6):(0.5-3).

3. The preparation method according to claim 1, characterized in that: Before the enzymatic hydrolysis, the ginkgo leaf raw material is mixed with water, and the solid-liquid ratio is 1:(8-15) g / mL; Preferably, the added amount of the complex enzyme is 0.1‰-0.5‰ of the mass of the ginkgo leaf raw material; Preferably, the enzymatic hydrolysis temperature is 40-60°C and the time is 0.5-2h.

4. The preparation method according to claim 1, characterized in that: In step S1, the solid-liquid ratio of the ginkgo leaf raw material to the ethanol aqueous solution is 1:(4-10) g / mL; Preferably, the volume percentage of ethanol in the ethanol aqueous solution in step S1 is 30%-90%; Preferably, the number of reflux extractions in step S1 is 2-5 times, and the time of reflux extraction is 1-3h / time; Preferably, the volume ratio of the concentrate to water in step S1 is 1:(2-20); Preferably, the temperature of mixing with water and heating in step S1 is 50-100°C and the time is 20-60min; Preferably, the standing temperature in step S1 is 2-6°C and the standing time is 18-48h.

5. The preparation method according to claim 1, characterized in that: The type of the macroporous resin in step S2 includes any one of AB-8, D101 or HPD100; Preferably, the enrichment in step S2 specifically comprises: applying the crude extract to a macroporous resin for adsorption, eluting with water, analyzing with a 40%-90% ethanol aqueous solution, and collecting the eluate to obtain; Preferably, the elution flow rate is 120-180 L / h.

6. The preparation method according to claim 1, characterized in that: The temperature of the concentrated drying in step S3 is 60-80°C; Preferably, in step S3, the solid-liquid ratio of the crude ginkgo leaf extract to the ethanol aqueous solution is 1:(4-10) g / mL; Preferably, the volume percentage of ethanol in the ethanol aqueous solution in step S3 is 40%-90%; Preferably, the temperature of the hot dipping in step S3 is 40-100°C and the time is 20-60min; Preferably, the cooling temperature in step S3 is 2-6°C and the cooling time is 18-48h.

7. The preparation method according to claim 1, characterized in that: The type of the macroporous resin in step S4 includes DM130; Preferably, the enrichment specifically comprises: loading the refined extract onto a polyamide resin and a macroporous resin in sequence for adsorption, washing with water to remove impurities, analyzing with a 40%-90% ethanol aqueous solution, and collecting the eluate to obtain; Preferably, the extract is further concentrated to remove alcohol, the concentrate is mixed with water, cooled and filtered to collect the filtrate before loading; Preferably, the volume ratio of the concentrate to water is 1:(10-40); Preferably, the cooling temperature is 2-6°C and the time is 18-48h; preferably, the elution flow rate of the polyamide resin is 80-150L / h; Preferably, the macroporous resin is adsorbed for 20-60 min at a flow rate of 60-100 L / h.

8. The preparation method according to claim 1, characterized in that: Step S5: the second eluate is further concentrated and alcohol-removed before being allowed to stand; Preferably, the temperature of the standing still in step S5 is 2-6°C and the time is 18-48h; Preferably, the flocculant in step S5 comprises a combination of gelatin, chitosan and guar gum; Preferably, the mass ratio of gelatin, chitosan and guar gum is (0.5-2):1:(1-3); Preferably, the amount of the flocculant in step S5 is 0.05‰-0.1‰ of the weight of the filtrate; Preferably, the mixing temperature in step S5 is 50-60° C. and the mixing time is 10-30 min.

9. A ginkgo leaf extract, characterized in that The ginkgo leaf extract is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the ginkgo leaf extract according to claim 9 in the preparation of moisturizing and / or antioxidant products.

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