CN108904343B - A ginkgo leaf composition for delaying aging and its preparation method and application - Google Patents

Abstract

The present invention relates to a cosmetic efficacy additive, specifically providing a ginkgo leaf composition with an anti-aging effect, wherein the composition contains 50-95 parts of ginkgo leaf total lactones, 22-60 parts of ginkgo leaf total flavonoids, and the ginkgo leaf ginkgolic acid is less than 0.0035 parts by weight, wherein the ginkgo leaf total lactones are selected from one or more of ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J and bilobalide, and the ginkgo leaf total flavonoids are selected from one or more of rutin, quercetin-3-O-glucoside, kaempferol-3-O-glucoside, isorhamnetin-3-O-glucoside and myricetin. The composition can be obtained by mixing monomers, or by extracting and separating and purifying ginkgo leaves, and step-by-step purification by different resins can effectively enrich the effective ingredients in ginkgo leaves, significantly improve the effect of the composition in delaying cell aging, and can be widely used in various cosmetics.

Description

A ginkgo leaf composition for delaying aging and its preparation method and application

Technical Field

The invention belongs to cosmetic additives, and relates to a ginkgo leaf composition, an extraction method and application thereof, and specifically relates to a ginkgo leaf composition for delaying aging, an extraction method and application thereof.

Background Art

Ginkgo biloba appeared on the earth as early as 345 million years ago. It is a relict tree species unique to China during the Quaternary Ice Age. 70% of the resources of Ginkgo biloba are distributed in my country. More than 50 countries in the world use Ginkgo biloba extract as a source of plant raw materials for the treatment of cardiovascular and cerebrovascular diseases.

The chemical composition of Ginkgo biloba is quite complex. The main active ingredients in Ginkgo biloba are divided into three categories: flavonoids, ginkgolides and ginkgolic acid. At present, more than 34 flavonoids have been isolated from Ginkgo biloba, and more than 70 flavonoids have been preliminarily identified by mass spectrometry. The main flavonoid aglycones in Ginkgo biloba are quercetin, kaempferol, derivatives of isorhamnetin, some glucose and rhamnose connected in different forms and quantities, and some biflavonoid compounds formed by the polymerization of two molecules of flavonoid mother nuclei through C-C bonds. There are also some characteristic lactone components in Ginkgo biloba, including diterpenoid lactone compounds and sesquiterpenoid lactone compounds. In addition, ginkgolic acid is the main toxic substance in Ginkgo biloba extract, which has sensitization, embryotoxicity, cytotoxicity, mutagenicity and immunotoxicity. At present, the EGB production standard in the Chinese Pharmacopoeia requires that the content of ginkgolic acid be controlled below 10ppm, and the EGB production standard in the European and American Pharmacopoeias requires that the content of ginkgolic acid be controlled below 5ppm.

Ginkgo biloba, as a plant with both medicinal and edible properties, has a variety of biological activities. In the 1960s, there were studies on the pharmacological effects and practical applications of ginkgo biloba. The first ginkgo biloba extract EGB761 developed by Dr. Weimar Shupe Pharmaceuticals in Germany has a significant therapeutic effect on cardiovascular and cerebrovascular diseases. The active ingredients of ginkgo biloba extract have the effects of treating nervous system diseases, lowering blood lipids, improving the body's immunity, anti-oxidation and scavenging free radical activity, and have good medicinal value and health care effects. In addition, EGB also has the effect of inhibiting Staphylococcus aureus, streptococci and fungi. Modern pharmacological studies have shown that ginkgo biloba extract has the effects of anti-platelet activation factor, anti-cerebral ischemia and hypoxia, lowering blood lipids, scavenging free radicals, relaxing bronchial smooth muscles, anti-inflammatory and enhancing nervous system activity, and has significant preventive and therapeutic effects on coronary heart disease, hypertension, angina pectoris, arteriosclerosis, cerebral dysfunction, senile dementia, memory loss, aging and other diseases related to cardiovascular and cerebrovascular circulation. At present, ginkgo leaf extract has been widely used in drugs for treating cardiovascular and cerebrovascular diseases. For example, patent application CN103463145A discloses a refined ginkgo leaf extract made from fallen ginkgo leaves, which has excellent antioxidant, anti-platelet aggregation and neuronal cell protection effects, and has low adverse reactions. It can be used as the main raw material for drugs and health foods for preventing and treating cardiovascular and cerebrovascular diseases; patent application CN106420850A discloses a ginkgo leaf composition, which contains 25-43% total flavonol glycosides, 6.5-17% ginkgolides, 3-5% bilobalide, and less than 3 ppm ginkgolic acid, and has a good therapeutic effect in treating cardiovascular diseases.

In recent years, studies have shown that Ginkgo biloba has a good protective effect on the skin, which is specifically reflected in inhibiting UVB radiation and photoaging, promoting blood microcirculation, anti-oxidation and inhibiting cell damage, anti-inflammatory and neuroprotection, and has a large application space in cosmetics. Patent application CN107913204A discloses a Ginkgo biloba skin care lotion, the raw materials and weight ratios in its formula are: 10 parts of Ginkgo biloba extract, 3 parts of stearic acid monoglyceride, 3 parts of white mineral oil, 3 parts of caprylic acid/capric acid triglyceride, 1 part of hydrogenated vegetable oil, 2.5 parts of stearic acid, 10 parts of polyacrylic acid resin, 2.5 parts of sorbitol, 3 parts of propylene glycol, 0.7 parts of triethanolamine, 0.2 parts of methyl paraben, an appropriate amount of essence, an appropriate amount of preservatives, and deionized water added to 100 parts. The Ginkgo biloba skin care lotion prepared by it has the effects of anti-wrinkle and whitening, scavenging free radicals, etc. Patent application CN103387896A discloses a ginkgo leaf whitening compound soap. The flavonoids and amino acid-synthesized collagen components in ginkgo leaves play a significant role in human beauty, inhibiting melanin growth, and maintaining skin luster and elasticity. The above patents do not pay attention to the efficacy of ginkgo leaves in anti-cell aging. At the same time, the specific components of ginkgo leaf extracts and how to inhibit cell aging have not been studied, and the safety of the product cannot be guaranteed.

At present, a large number of studies have shown that ginkgo biloba flavonoids can play a protective role against photoaging by inhibiting UVB radiation. The main effects of ginkgolides are promoting blood microcirculation, anti-oxidation, inhibiting cell damage, anti-inflammation and neuroprotection. For example, Jiang Xiuxin ^[58] found through research that ginkgolide B can significantly improve the platelet function of the elderly. It is not only a platelet activating factor (PAF) antagonist, but also has the effect of reducing ROS production. However, the physiological effects of ginkgo leaf extract are closely related to its complex chemical composition. The research on its active ingredients at home and abroad mainly focuses on ginkgolide A, B, J and bilobalide, ginkgo biloba flavonoids and isoginkgo biloba flavonoids. The main efficacy research focuses on inhibiting cell damage, anti-inflammatory and antioxidant, neuroprotection and promoting blood circulation. However, there is no in-depth study on the structure-activity relationship between the effective ingredients and the activity. In addition, based on the research on its efficacy at home and abroad, ginkgo leaf, as a plant source for the treatment of cardiovascular and cerebrovascular diseases, has good prospects in delaying aging, but there is currently no in-depth research on its mechanism of action in delaying aging.

Summary of the invention

Based on the defects of the above-mentioned prior art, the present invention provides a ginkgo leaf composition with anti-aging effect. By screening various active ingredients in ginkgo leaves and clarifying the composition of the composition, the prepared composition has good anti-aging effect, can inhibit the expression of MMP-1 protein, and promote the expression of Collagen I in fibroblasts.

The invention provides a ginkgo leaf composition with anti-aging effect, which contains 50-95 parts of ginkgo leaf total lactones, 22-60 parts of ginkgo leaf total flavonoids, and the content of ginkgo acid in the ginkgo leaf is less than 0.0035 parts.

Furthermore, the ginkgo leaf composition contains 70-90 parts of ginkgo leaf total lactones, 28-45 parts of ginkgo leaf total flavonoids, and the content of ginkgo acid in ginkgo leaf is less than 0.0030 parts.

Furthermore, the ginkgo leaf composition contains 72-78 parts of total ginkgo leaf lactones, 34-44 parts of total ginkgo leaf flavonoids, and the content of ginkgo acid in ginkgo leaf is less than 0.0028 parts.

Furthermore, the total lactones of Ginkgo biloba leaves are selected from one or more of Ginkgolide A, Ginkgolide B, Ginkgolide C, Ginkgolide J and Bilobalide.

Furthermore, the total lactones of Ginkgo biloba leaves are selected from one or more of 9-15 parts of Ginkgolide A, 5-10 parts of Ginkgolide B, 7-12 parts of Ginkgolide C, 1-8 parts of Ginkgolide J and 48-55 parts of Bilobalide in parts by weight.

Furthermore, the total flavonoids of ginkgo leaves are selected from one or more of rutin, quercetin-3-O-glucoside, kaempferol-3-O-glucoside, isorhamnetin-3-O-glucoside and myricetin.

Furthermore, the total flavonoids of ginkgo leaves are selected from one or more of 12-18 parts of rutin, 3-8 parts of quercetin-3-O-glucoside, 1-5 parts of kaempferol-3-O-glucoside, 1-5 parts of isorhamnetin-3-O-glucoside and 15-24 parts of myricetin in terms of weight.

Furthermore, the ginkgo leaf composition comprises, by weight, 2-4 parts of kaempferol-3-O-glucoside, 2-4 parts of isorhamnetin-3-O-glucoside, 18-20 parts of myricetin, 10-12 parts of ginkgolide A and 48-52 parts of bilobalide.

Furthermore, the ginkgo leaf composition further comprises, by weight, 15-18 parts of rutin, 4-6 parts of quercetin-3-O-glucoside, 6-9 parts of ginkgolide B, 8-11 parts of ginkgolide C and 2-7 parts of ginkgolide J.

The composition of the present invention can be prepared by directly mixing the monomers, or can be obtained by extracting from ginkgo leaves.

The present invention further provides a method for preparing the above-mentioned ginkgo leaf composition, comprising the following steps:

(1) Ginkgo biloba leaves are dried and crushed, ethanol is added for ultrasonic extraction, and the supernatant is concentrated under reduced pressure to obtain a crude extract of ginkgo biloba leaves;

(2) diluting the crude extract of ginkgo leaves with water, centrifuging, and adsorbing the supernatant with a macroporous adsorption resin, using ethanol as an eluent to obtain a ginkgo leaf eluate;

(3) The eluate from the ginkgo leaves is adsorbed by a cationic adsorption resin and a macroporous adsorption resin in sequence, with ethanol as the eluent, and concentrated under reduced pressure to obtain the result.

Furthermore, the preparation method of the ginkgo leaf composition comprises the following steps:

(1) Ginkgo biloba leaves are dried and crushed, passed through a 80-120 mesh sieve, added with 12-18 times 90%-100% ethanol, and ultrasonically extracted for 0.5-1.5 hours, extracted 2-4 times, combined the supernatants and concentrated under reduced pressure at 40-55° C. to obtain a crude extract of ginkgo biloba leaves;

(2) diluting the crude extract of ginkgo leaves with 8-12 times of water, centrifuging, and adsorbing the supernatant with DM130 macroporous adsorption resin, using 55-65% ethanol as eluent to obtain a ginkgo leaf eluate;

(3) The eluate from the ginkgo leaves was adsorbed by 732 cationic adsorption resin and HPD5000 macroporous adsorption resin in turn, with 55-65% ethanol as the eluent, and concentrated under reduced pressure to obtain the result.

The present invention further provides the use of the ginkgo leaf composition in preparing an anti-aging cosmetic functional additive.

The beneficial effects of the present invention are:

(1) The ginkgo leaf composition provided by the present invention has good anti-aging activity. The composition can be prepared by mixing monomers of each component, or can be obtained by extracting ginkgo leaves. Kaempferol-3-o-glucoside, isorhamnetin-3-o-glucoside, myricetin, ginkgolide A and bilobalide in the composition can significantly inhibit the expression of MMP-1 protein in fibroblasts. Each component can play a good anti-aging effect when used alone. Ginkgolide A and bilobalide can further promote the expression of Collagen I protein in fibroblasts, and the anti-aging effect is more obvious. If these five components are used in combination, they can work synergistically to more significantly inhibit skin aging. Although ginkgolide B, ginkgolide C, ginkgolide J, rutin, and quercetin-3-O-glucoside have no significant effect in inhibiting the expression of MMP-1 protein in fibroblasts, they will not affect the anti-aging effects of kaempferol-3-o-glucoside, isorhamnetin-3-o-glucoside, myricetin, ginkgolide A, and bilobalide. At the same time, the presence of ginkgolide B, ginkgolide C, ginkgolide J, rutin, and quercetin-3-O-glucoside can further promote the anti-aging effect of the composition by improving the DPPH scavenging ability and other aspects.

(2) The composition of the present invention can also be obtained by extracting the active ingredients of Ginkgo biloba leaves, using ethanol as the eluent, respectively passing through 732 cationic adsorption resin and HPD5000 macroporous adsorption resin to enrich the active ingredients in the Ginkgo biloba extract, and the final Ginkgo biloba extract contains 50-95 parts of total Ginkgo biloba lactones, 22-60 parts of total Ginkgo biloba flavonoids, and the content of Ginkgo biloba ginkgolic acid is less than 0.0035 parts. The Ginkgo biloba extract extracted by this process has a high content and purity of active ingredients, and a simple preparation process. It has significant effects in inhibiting the expression of fibroblast MMP-1 protein and promoting the expression of Collagen I protein, and can be widely used in cosmetics to improve the anti-aging effect.

(3) The present invention studies the effects of the active ingredients of Ginkgo biloba extract, which are five characteristic lactone components, namely Ginkgolide A, Ginkgolide B, Ginkgolide C, Ginkgolide J and Bilobalide, and ten monomer components, namely rutin, quercetin-3-O-glucoside, kaempferol-3-O-glucoside, isorhamnetin-3-O-glucoside and myricetin, on the expression of fibroblast MMP-1 protein and Collagen I protein. The present invention clearly and thoroughly studies the composition and mechanism of the active ingredients of Ginkgo biloba extract in anti-aging, filling the gap in the study of the structure-activity relationship of the active ingredients of Ginkgo biloba extract in delaying aging.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the determination of the inhibitory activity of 10 monomer components on fibroblast MMP-1

Figure 2 shows the effects of 10 monomer components on the expression of Collagen I in fibroblasts

Among them, 1-rutin 2-quercetin-3-o-glucoside 3-kaempferol-3-o-glucoside

4—Isorhamnetin-3-o-glucoside 5—Myricetin 6—Ginkgolide A

7—Ginkgolide B 8—Ginkgolide C 9—Ginkgolide J

10—Bilobalide

DETAILED DESCRIPTION

Example 1 Ginkgo leaf composition and extraction method thereof

(1) Ginkgo biloba leaves were dried and crushed, passed through a 100-mesh sieve, and ultrasonically extracted for 1 h in 15-fold 95% ethanol for 3 times. The supernatants were combined and concentrated under reduced pressure at 50° C. to obtain a crude extract of ginkgo biloba leaves;

(2) The crude extract of ginkgo leaves was diluted with 10 times water and centrifuged, and the supernatant was adsorbed by DM130 macroporous adsorption resin, and 60% ethanol was used as eluent to obtain the ginkgo leaf eluate;

(3) The eluate from the ginkgo leaves was adsorbed by 732 cationic adsorption resin and HPD5000 macroporous adsorption resin in sequence, with 60% ethanol as the eluent, and concentrated under reduced pressure to obtain the product.

Active ingredients and content detection:

Weigh an appropriate amount of the above extracts for redissolution, and prepare it to a concentration of 1 mg/mL with methanol. Quantitative analysis of the lactone components, ginkgolic acid components, and flavonoid components of the active extracts was performed by UPLC/MS/MS and HPLC/DAD methods, as follows:

(1) Detection of lactone content:

ESI-mode: dry nitrogen flow rate 10L/min, temperature 35°C, nebulizer pressure: 30psi; capillary voltage: 3500V; MRM mode quantitative, the capillary outlet voltage and collision energy of each reference sample were optimized to obtain more stable ions and higher response.

Chromatographic conditions: ACQUITY UPLC BEH C18 1.7 μm column, mobile phase of acetonitrile and 0.1% formic acid solution, gradient elution (0-0.5 min, 10% A, 0.5-4 min, 90% A), flow rate 0.3 ml/min, column temperature 30° C., injection volume 2 μl.

Drawing of standard curve: Accurately measure appropriate amount of Ginkgolide A, B, C, J and biloba lactone standard reference substances, place them in volumetric flasks, add methanol to prepare mixed reference substance solutions containing single reference substances with mass concentrations of 0.008, 0.008, 0.008, 0.0015, and 0.050 mg/ml, respectively. Change the concentration of the mixed reference substance solution by changing the injection volume, analyze the prepared mixed reference substance according to the above-mentioned chromatographic conditions, and draw the standard curve.

Sample determination: Weigh an appropriate amount of the above extract and re-dissolve it in methanol to a concentration of 1 mg/mL, and analyze it according to the above chromatographic conditions.

(2) Flavonoid content detection:

Chromatographic conditions: mobile phase is acetonitrile and 0.2% phosphoric acid solution, flow rate is 0.5ml/min, column temperature is 30℃, injection volume is 5μl, detection wavelength is 270nm, gradient elution is as follows:

Drawing of standard curve: Accurately weigh appropriate amount of rutin, quercetin-3-O-glucoside, kaempferol-3-O-glucoside, isorhamnetin-3-O-glucoside, and myricetin reference substances, add methanol to prepare mixed reference substance solutions containing single reference substances with mass concentrations of 0.012, 0.006, 0.012, 0.012, and 0.012, respectively, and dilute the mixed reference substance solutions with methanol to different concentrations, perform detection according to the above-mentioned chromatographic conditions, and draw the standard curve.

Sample determination: Weigh an appropriate amount of the above extract for re-dissolution, prepare it to a concentration of 1 mg/mL with methanol, filter it through a 0.22 μm microporous filter membrane, and then detect it according to the above liquid phase conditions.

(3) Detection of Ginkgo acid content

Mass spectrometry conditions: ESI-mode: capillary voltage: 2500 V, desolvation gas flow rate 800 L/H, desolvation gas temperature 450°C, source temperature 150°C, nebulizer pressure: 30 psi; MRM mode, the capillary outlet voltage and collision energy of each reference substance were optimized to obtain more stable ions and higher responses.

Chromatographic conditions: ACQUITY UPLC BEH C18 1.7 μm column, mobile phase of acetonitrile and 0.1% formic acid solution, gradient elution (0-1.0 min, 10% A, 1.0-6.0 min, 90% A), flow rate of 0.4 ml/min, column temperature of 35°C, sample room temperature of 10°C, injection volume of 10 μl.

Drawing of standard curve: Accurately weigh appropriate amount of Ginkgo biloba C13:0, C15:0, C15:1, C17:1, and C17:2 reference substances, prepare a mixed solution of 2 mg/ml with methanol, dilute it step by step with methanol, enter liquid chromatography for detection, and draw a standard curve.

Sample determination: Weigh an appropriate amount of the above extract for re-dissolution, prepare it to a concentration of 1 mg/mL with methanol, filter it through a 0.22 μm microporous filter membrane, and enter it into a liquid chromatograph for determination. The results are as follows.

Lactones 7.94% Flavonoids 4.36% Ginkgolic acid 2.69ppm Ginkgolide A 0.97% Rutin 1.61% C13:0 0.09ppm Ginkgolide B 0.66% Quercetin-3-O-glucoside 0.43% C15:0 0.06ppm Ginkgolide C 0.85% Kaempferol-3-O-glucoside 0.20% C15:1 0.23ppm Ginkgolide J 0.25% Isorhamnetin-3-O-glucoside 0.31% C17:1 2.32ppm Bilobalide 5.21% Myricetin 1.81%

Example 2 Ginkgo leaf composition and extraction method thereof

(1) Ginkgo biloba leaves were dried and crushed, passed through an 80-mesh sieve, and ultrasonically extracted for 1.5 h with 12-fold 90% ethanol. The extraction was repeated twice, and the supernatants were combined and concentrated under reduced pressure at 40° C. to obtain a crude extract of ginkgo biloba leaves;

(2) The crude extract of ginkgo leaves was diluted with 8 times water and centrifuged, and the supernatant was adsorbed by DM130 macroporous adsorption resin, and 55% ethanol was used as the eluent to obtain the ginkgo leaf eluate;

(3) The eluate from the ginkgo leaves was adsorbed by 732 cationic adsorption resin and HPD5000 macroporous adsorption resin in turn, with 55% ethanol as the eluent, and concentrated under reduced pressure to obtain the result.

Active ingredient and content detection: Same as Example 1, the results are as follows.

Lactones 7.26% Flavonoids 3.4% Ginkgolic acid 3.21ppm Ginkgolide A 0.92% Rutin 1.24% C13:0 0.12ppm Ginkgolide B 0.51% Quercetin-3-O-glucoside 0.33% C15:0 0.13ppm Ginkgolide C 0.77% Kaempferol-3-O-glucoside 0.17% C15:1 0.20ppm Ginkgolide J 0.13% Isorhamnetin-3-O-glucoside 0.12% C17:1 2.76ppm Bilobalide 4.93% Myricetin 1.54%

Example 3 Ginkgo leaf composition and extraction method thereof

(1) Ginkgo biloba leaves were dried and crushed, passed through a 120-mesh sieve, and ultrasonically extracted for 0.5 h with 18-fold 100% ethanol for 4 times. The supernatants were combined and concentrated under reduced pressure at 55° C. to obtain a crude extract of ginkgo biloba leaves;

(2) The crude extract of ginkgo leaves was diluted with 8 times water and centrifuged, and the supernatant was adsorbed by DM130 macroporous adsorption resin, and 65% ethanol was used as eluent to obtain the ginkgo leaf eluate;

(3) The eluate from the ginkgo leaves was adsorbed by 732 cationic adsorption resin and HPD5000 macroporous adsorption resin in sequence, with 65% ethanol as the eluent, and concentrated under reduced pressure to obtain the result.

Active ingredient and content detection: Same as Example 1, the results are shown in the following table.

Lactones 9.33% Flavonoids 6.01% Ginkgo acid 2.28ppm Ginkgolide A 1.18% Rutin 1.85% C13:0 0.09ppm Ginkgolide B 0.87% Quercetin-3-O-glucoside 0.82% C15:0 0.12ppm Ginkgolide C 1.11% Kaempferol-3-O-glucoside 0.47% C15:1 0.09ppm Ginkgolide J 0.69% Isorhamnetin-3-O-glucoside 0.41% C17:1 1.98ppm Bilobalide 5.48% Myricetin 2.46%

Example 4-6 Ginkgo biloba leaf composition

The 10 monomers of ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, bilobalide, rutin, quercetin-3-O-glucoside, kaempferol-3-O-glucoside, isorhamnetin-3-O-glucoside and myricetin were mixed according to the following weight parts, as shown in the following table:

Comparative Example 1 Ginkgo leaf compositions obtained by different adsorption resin treatment processes

Except that AB-8 macroporous resin is used in step (2), the rest is the same as in Example 1, as follows:

(1) Ginkgo biloba leaves were dried and crushed, passed through a 100-mesh sieve, and ultrasonically extracted for 1 h in 15-fold 95% ethanol for 3 times. The supernatants were combined and concentrated under reduced pressure at 50° C. to obtain a crude extract of ginkgo biloba leaves;

(2) The crude extract of ginkgo leaves was diluted with 10 times water and centrifuged, and the supernatant was adsorbed by AB-8 macroporous adsorption resin, and 60% ethanol was used as eluent to obtain the ginkgo leaf eluate;

(3) The eluate from the ginkgo leaves was adsorbed by 732 cationic adsorption resin and HPD5000 macroporous adsorption resin in sequence, with 60% ethanol as the eluent, and concentrated under reduced pressure to obtain the product.

Active ingredient and content detection: Same as Example 1, the results are as follows.

Comparative Example 2 Ginkgo leaf compositions obtained by different adsorption resin treatment processes

Except that D101 macroporous resin is used in step (3), the rest is the same as in Example 1, as follows:

(1) Ginkgo biloba leaves were dried and crushed, passed through a 100-mesh sieve, and ultrasonically extracted for 1 h in 15-fold 95% ethanol for 3 times. The supernatants were combined and concentrated under reduced pressure at 50° C. to obtain a crude extract of ginkgo biloba leaves;

(2) The crude extract of ginkgo leaves was diluted with 10 times water and centrifuged, and the supernatant was adsorbed by DM130 macroporous adsorption resin, and 60% ethanol was used as eluent to obtain the ginkgo leaf eluate;

(3) The eluate from the ginkgo leaves was adsorbed with 732 cationic adsorption resin and D101 macroporous adsorption resin in turn, with 60% ethanol as the eluent, and concentrated under reduced pressure to obtain the result.

Active ingredient and content detection: Same as Example 1, the results are as follows.

Lactones 5.28% Flavonoids 9.61% Ginkgo acid 4.20ppm Ginkgolide A 0.65% Rutin 3.07% C13:0 0.69ppm Ginkgolide B 0.26% Quercetin-3-O-glucoside 1.65% C15:0 0.22ppm Ginkgolide C 0.87% Kaempferol-3-O-glucoside 1.97% C15:1 0.42ppm Ginkgolide J 0.43% Isorhamnetin-3-O-glucoside 0.84% C17:1 2.87ppm Bilobalide 3.07% Myricetin 2.08%

Comparative Example 3 Ginkgo leaf compositions obtained by different extraction processes

The crude extract of ginkgo leaves prepared in step 1 of Example 1 was separated by organic solvent extraction to obtain a composition of different extraction parts of ginkgo leaves, and the rest was the same as in Example 1, specifically as follows:

(1) Ginkgo biloba leaves were dried and crushed, passed through a 100-mesh sieve, and ultrasonically extracted for 1 h in 15-fold 95% ethanol for 3 times. The supernatants were combined and concentrated under reduced pressure at 50° C. to obtain a crude extract of ginkgo biloba leaves;

(2) The crude extract of ginkgo leaves obtained above was extracted with petroleum ether, ethyl acetate and n-butanol in sequence, and the extracts of the three parts obtained by extraction were first concentrated by a rotary evaporator, and then the concentrated solution was dried in a vacuum drying oven to finally obtain ginkgo leaf extracts of the three extraction parts, namely, petroleum ether, ethyl acetate and n-butanol extracts.

Active ingredient and content detection: Same as Example 1, the results are as follows.

Petroleum ether part:

Lactones 3.26% Flavonoids 2.09% Ginkgo acid 3.07ppm Ginkgolide A 0.12% Rutin 0.25% C13:0 0.09ppm Ginkgolide B 0.26% Quercetin-3-O-glucoside 0.82% C15:0 0.12ppm Ginkgolide C 0.64% Kaempferol-3-O-glucoside 0.38% C15:1 0.88ppm Ginkgolide J 0.98% Isorhamnetin-3-O-glucoside 0.18% C17:1 1.98ppm Bilobalide 1.26% Myricetin 0.46%

Ethyl acetate part

Lactones 10.89% Flavonoids 7.36% Ginkgo acid 4.24ppm Ginkgolide A 0.83% Rutin 1.25% C13:0 0.11ppm Ginkgolide B 0.26% Quercetin-3-O-glucoside 1.22% C15:0 0.54ppm Ginkgolide C 0.74% Kaempferol-3-O-glucoside 1.18% C15:1 1.13ppm Ginkgolide J 1.11% Isorhamnetin-3-O-glucoside 1.48% C17:1 2.46ppm Bilobalide 7.95% Myricetin 2.23%

n-Butanol part

Lactones 4.76% Flavonoids 3.25% Ginkgo acid 1.98ppm Ginkgolide A 1.12% Rutin 0.27% C13:0 0.05ppm Ginkgolide B 0.37% Quercetin-3-O-glucoside 0.87% C15:0 0.07ppm Ginkgolide C 0.65% Kaempferol-3-O-glucoside 0.44% C15:1 0.11ppm Ginkgolide J 0.85% Isorhamnetin-3-O-glucoside 0.68% C17:1 1.75ppm Bilobalide 1.77% Myricetin 0.99%

Comparative Example 4 Ginkgo leaf compositions obtained by different elution processes

Except that the eluent in steps (2) and (3) is 95% ethanol, the rest is the same as in Example 1, as follows:

(1) Ginkgo biloba leaves were dried and crushed, passed through a 100-mesh sieve, and ultrasonically extracted for 1 h in 15-fold 95% ethanol for 3 times. The supernatants were combined and concentrated under reduced pressure at 50° C. to obtain a crude extract of ginkgo biloba leaves;

(2) The crude extract of ginkgo leaves was diluted with 10 times water and centrifuged, and the supernatant was adsorbed by DM130 macroporous adsorption resin, and 95% ethanol was used as eluent to obtain the ginkgo leaf eluate;

(3) The eluate from the ginkgo leaves was adsorbed by 732 cationic adsorption resin and HPD5000 macroporous adsorption resin in turn, with 95% ethanol as the eluent, and concentrated under reduced pressure to obtain the product.

Comparative Examples 5-7 Ginkgo Leaf Composition

The monomers were mixed according to the following weight parts, as shown in the following table:

Compared with Example 1, Comparative Example 5 does not contain Ginkgolide B, but contains 9.7 parts by weight of Ginkgolide M; Comparative Example 6 does not contain rutin, but contains 16.1 parts by weight of bilobalol; Comparative Example 7 does not contain Ginkgolide J and quercetin-3-O-glucoside, but contains 2.5 parts by weight of Ginkgolide M and 4.3 parts by weight of apigenin-7-glucoside.

Test Example 1 Detection of anti-aging activity of Ginkgo biloba leaf composition and active ingredients

1.1 Fibroblast viability assay:

The ten monomer components, including ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, bilobalide, rutin, quercetin-3-O-glucoside, kaempferol-3-O-glucoside, isorhamnetin-3-O-glucoside and myricetin, were prepared with 1% DMSO solution into solutions with concentrations of 0.4, 0.2, 0.1, and 0.05 mg/mL, respectively, and a negative control group was set up.

Fibroblasts were plated in a 96-well plate at a density of 1×10 ⁵ /mL, and 0.1mL of cell suspension was added to each well. After the cells adhered to the wall, drugs were added. After 24 hours of drug action, 110μL of CCK-8 reagent diluted 1:10 with serum-free medium was added for 1 hour, and its absorbance was measured at 450nm on an ELISA reader. Cell survival rate was calculated according to the following formula.

Cell survival rate = (absorbance of each group/absorbance of blank group) × 100%

The results are as follows:

Table 1 Effects of monomer components on cell viability of fibroblasts (n=3, )

As shown in the table above, at a concentration of 0.4 mg/mL, the cell survival rates of isorhamnetin-3-o-glucoside and myricetin were (63.86±0.7)% and (58.58±4.62)%, respectively, and there was a certain degree of cytotoxicity. At a concentration of 0.2 mg/mL, the cell survival rates reached more than 80%;

In addition, at a concentration of 0.4 mg/mL, the cell viability of rutin, quercetin-3-o-glucoside, kaempferol-3-o-glucoside, ginkgolide A, B, C, J and bilobalide all reached more than 80%. Therefore, based on the actual situation, 0.2 mg/mL and 0.1 mg/mL were selected as the experimental concentrations for the subsequent MMP-1 index detection.

1.2 MMP-1 protein expression detection:

Fibroblasts were plated in a 6-well plate at a density of 2×10 ⁴ /mL, and 1 mL of cell suspension was added to each well. After the cells adhered to the wall, the drug was added. After 24 hours of drug action, the supernatant of each group of cells was taken and centrifuged at 1000 rpm for 15 minutes. After centrifugation, the supernatant was taken for testing. The test was performed according to the operating procedures of the MMP-1 detection kit. The standard curve was fitted first, and the content of MMP-1 in the sample was calculated on this basis. The results are shown in Figure 1.

The results showed that kaempferol-3-o-glucoside, isorhamnetin-3-o-glucoside, myricetin, ginkgolide A and bilobalide could inhibit the expression of MMP-1, and compared with the blank control group, the effect of bilobalide on MMP-1 was significantly different (P < 0.05), and the effects of kaempferol-3-o-glucoside, isorhamnetin-3-o-glucoside, myricetin and ginkgolide A on MMP-1 were very significantly different (P < 0.01).

1.3 Effect on Collagen I protein expression

Fibroblasts were plated in a 6-well plate at a density of 2×10 ⁴ /mL, and 1mL of cell suspension was added to each well. After the cells adhered to the wall, the drug was added. After 24 hours of drug action, the supernatant of each group of cells was taken and centrifuged at 1000 rpm for 15 minutes. After centrifugation, the supernatant was taken for testing. The test was performed according to the operating procedures of the Collagen I detection kit. The standard curve was fitted first, and the content of Collagen I in the sample was calculated on this basis. The results are shown in Figure 2.

The results showed that compared with the control group, bilobalide significantly promoted the expression of Collagen I in fibroblasts (P < 0.05), and ginkgolide A had a very significant promoting effect on the expression of Collagen I in fibroblasts (P < 0.01).

1.4 Study on the antioxidant activity of Ginkgo biloba leaf composition

The determination method of the effect of Ginkgo biloba extract on fibroblast cell viability, fibroblast MMP-1 inhibitory activity, and Collagen I protein expression is the same as 1.1-1.3.

Among them, the ginkgo leaf extracts prepared in different embodiments and comparative examples were dissolved in anhydrous ethanol and prepared into 1, 0.5, 0.25, and 0.125 mg/mL; a solvent control group and a negative control group were set up to carry out fibroblast cell viability assay, and then the required concentration for subsequent experiments was determined according to the results of the fibroblast cell viability assay.

Table 2 Effects of different Ginkgo biloba leaf compositions on fibroblast cell viability (n=3, )

At concentrations below 0.5 mg/ml, all Ginkgo biloba leaf compositions were able to satisfy the requirement that the cell viability of fibroblasts reached more than 80%. To reduce costs and improve detection sensitivity, concentrations of 0.02 and 0.01 mg/ml were selected for subsequent experiments.

Table 3 Effect of Ginkgo biloba leaf composition on the secretion of MMP-1 by fibroblasts (n=3, )

Note: Compared with the negative control group, *P＜0.05, **P＜0.01.

It can be seen from the data in Table 3 that the effect of the ginkgo leaf composition provided by the present invention in inhibiting the expression of MMP-1 protein is significantly higher than that of comparative examples 1-7, indicating that the ginkgo leaf composition provided by the present invention has stronger biological activity, and changes in the composition ratio or composition will significantly affect its anti-aging effect.

Table 4 Effect of Ginkgo biloba leaf composition on the expression of collagen I protein in fibroblasts

Note: Compared with the negative control group, *P＜0.05, **P＜0.01.

It can be seen from the data in Table 4 that the effect of the ginkgo leaf composition provided by the present invention in promoting the expression of Collagen I protein is significantly higher than that of Comparative Examples 1-7, which further illustrates that the ginkgo leaf composition provided by the present invention has stronger biological activity, and changes in the composition ratio or composition will significantly affect its anti-aging effect.

The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims (3)

1. A ginkgo leaf composition with anti-aging effect, characterized in that the ginkgo leaf composition consists of ginkgo leaf total lactones and ginkgo leaf total flavonoids; The total lactones and total flavonoids of ginkgo leaves are composed of the following monomers by weight: 9.7 parts of ginkgolide A, 52.1 parts of bilobalide, 2 parts of kaempferol-3-O-glucoside, 3 parts of isorhamnetin-3-O-glucoside and 18.1 parts of myricetin, or 12 parts of ginkgolide A, 40 parts of bilobalide, 4 parts of kaempferol-3-O-glucoside, 4 parts of isorhamnetin-3-O-glucoside and 20 parts of myricetin, or 11 parts of ginkgolide A, 6 parts of ginkgolide B, 10 parts of ginkgolide C, 5 parts of ginkgolide J, 46 parts of bilobalide, 15 parts of rutin, 6 parts of quercetin-3-O-glucoside, 3 parts of kaempferol-3-O-glucoside, 2 parts of isorhamnetin-3-O-glucoside and 19 parts of myricetin, or 6.6 parts of ginkgolide B, 8.5 parts of ginkgolide C, 2.5 parts of ginkgolide J, 52.1 parts of bilobalide, 16.1 parts of rutin, 4.3 parts of quercetin-3-O-glucoside, 2 parts of kaempferol-3-O-glucoside, 3 parts of isorhamnetin-3-O-glucoside and 18.1 parts of myricetin, or 9.7 parts of ginkgolide A, 6.6 parts of ginkgolide B, 8.5 parts of ginkgolide C, 2.5 parts of ginkgolide J, 52.1 parts of bilobalide, 16.1 parts of rutin, 4.3 parts of quercetin-3-O-glucoside, 3 parts of isorhamnetin-3-O-glucoside and 18.1 parts of myricetin, or 9.7 parts of ginkgolide A, 6.6 parts of ginkgolide B, 8.5 parts of ginkgolide C, 2.5 parts of ginkgolide J, 52.1 parts of bilobalide, 16.1 parts of rutin, 4.3 parts of quercetin-3-O-glucoside, 2 parts of kaempferol-3-O-glucoside and 18.1 parts of myricetin. 2. The method for preparing the ginkgo leaf composition as claimed in claim 1, comprising the following steps: mixing the monomers of each component according to a certain proportion. 3. Use of the ginkgo leaf composition according to claim 1 in the preparation of an anti-aging cosmetic additive.