Ginseng composition with anti-wrinkle effect and preparation method and application thereof

Abstract

The present invention discloses a ginseng composition with anti-wrinkle efficacy, a preparation method and application thereof, and relates to the technical field of skin care products. The ginseng composition provided by the present invention includes 1 to 2 parts by mass of ginseng root extract, ginseng fruit extract and ginseng essential oil, and each component is extracted from ginseng, so that ginseng is fully utilized. At the same time, the scavenging rate of the composition for DPPH is as high as 55.14±1.42% or more, which is significantly better than a single component or a formula replaced by other similar components, showing a significant ability to scavenge free radicals and excellent antioxidant potential. In the anti-wrinkle efficacy test, the inhibition rate of zebrafish tail fin wrinkling is as high as 53.17%, which has excellent anti-wrinkle efficacy.

Description

Technical Field

[0001] The present invention relates to the technical field of skin care products, and in particular to a ginseng composition with anti-wrinkle effect, a preparation method and application thereof. Background Art

[0002] In the field of natural product research and application, ginseng, as a traditional Chinese medicinal material with a long history, has long been widely recognized for its medicinal value and health-care functions. Different parts of ginseng, including roots, fruits, and ginseng essential oil extracted through a specific process, are rich in bioactive ingredients, such as ginsenosides, polysaccharides, volatile oils, amino acids, and trace elements. These ingredients have shown significant beneficial effects in anti-oxidation, anti-inflammation, immunomodulation, anti-fatigue, and improvement of cardiovascular health, especially their potential in anti-oxidation and anti-aging. Therefore, various ginseng-related extracts have received widespread attention in the field of skin care products.

[0003] However, although ginseng extracts from different parts have certain effects in anti-oxidation, their effects are often limited by the singleness of ingredients and mechanisms of action. Existing related skin care products generally achieve anti-oxidation and anti-aging effects by removing free radicals in the human body, but free radicals are a general term for a class of highly active chemical species. Chemical species with unpaired electrons in their molecules are all free radicals, and there are tens of thousands of chemical reaction processes in the human body, and free radicals may be produced in various reaction processes. Therefore, the human body contains a wealth of different types of free radicals. The reason why a single ginseng extract has certain limitations in its antioxidant effect is that it cannot fully cover and effectively remove a variety of free radicals, resulting in the effect being often not significant enough.

[0004] In order to make the best use of ginseng and fully utilize its value, how to combine ginseng extracts from different parts to obtain skin care products with better anti-wrinkle effects still needs to be studied. Summary of the invention

[0005] In order to address the deficiencies of the prior art, the present invention provides a ginseng composition with anti-wrinkle efficacy, which comprises a ginseng root extract prepared by a specific method, a ginseng fruit extract and ginseng essential oil in a specific proportion, wherein the three different extracts contain different types of ginsenosides. Through this combination, the present invention can achieve comprehensive coverage and removal of various free radicals that cause oxidation and aging, and has excellent anti-wrinkle efficacy.

[0006] Another object of the present invention is to provide a method for preparing a ginseng composition having anti-wrinkle effects.

[0007] Another object of the present invention is to provide an application of a ginseng composition having anti-wrinkle effect.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] A ginseng composition with anti-wrinkle effect, comprising the following components calculated by weight:

[0010] 1-2 parts of ginseng root extract, 1-2 parts of ginseng fruit extract, 1-2 parts of ginseng essential oil;

[0011] The preparation method of the ginseng root extract comprises the following steps:

[0012] Ginseng roots and rhizomes are crushed in a mass ratio of 1:(1-3), and water-extracted twice at 90-100°C, with the solid-liquid ratio of the first water extraction being 1:(9-11) and the solid-liquid ratio of the second water extraction being 1:(7-9).

[0013] Specifically, ginseng root extract is prepared by extracting (usually water extraction) the root of ginseng, a plant of the Araliaceae family, and has significant pharmacological activity and wide application value. The main active ingredient of ginseng root extract is ginsenosides, with a total content of about 14.1%, including Rb1 (~4.17%), Rc (~4.25%), Rb2 (~3.57%), Rb3 (~0.37%) and Rd (~1.69%) and other key saponins.

[0014] Ginseng (PANAX GINSENG) fruit extract (i.e. ginseng seeds, ginseng fruit) is a plant active ingredient extracted from ginseng fruit, which is also rich in ginsenoside compounds, including prototype saponins (such as Rg1, Re, Rf, Rb1, Rc, Rb2, Rb3, Rd, etc.) and rare saponins (such as S-Rg3, R-Rg3, S-Rh1, R-Rh2, F4, Rh4, Rk1, etc.). Among them, Re has the highest content of ~30.74%, followed by Rd ​​(~17.97%) and Rc (~6.39%). These ginsenosides are the main active ingredients of ginseng fruit and have high medicinal and health value.

[0015] Ginseng essential oil is a complex plant compound extracted from ginseng mainly by supercritical extraction or distillation (compared with ginseng root extract, ginseng essential oil focuses more on extracting volatile oil components). Its components contain a variety of active substances such as ginsenosides, including 16 major ginsenosides: Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1, 20(S)-Rg3, 20(R)-Rg3, Rg5, Rg6, Rh4, Rk1, Rk3 and F4. The inventors of the present application have found through a large number of experimental verifications that the content of some ginsenosides in ginseng essential oil is relatively significant, such as Rb1 (~0.704 mg / g), Rc (~0.728 mg / g), (S)-Rg3 (~0.756 mg / g) and Rk1 (~0.906 mg / g), while other components such as Rg6 and Rh4 have not been detected. In addition, ginseng essential oil may also contain small amounts of other active ingredients, such as fatty acids, terpenoids, etc. These ingredients work together on the skin to provide comprehensive nourishment and protection for the skin.

[0016] In summary, ginseng root extract, ginseng fruit extract and ginseng essential oil have their own characteristics and significant differences in composition. These differences are not only reflected in the types and structures of the main active ingredients, but also in their unique biological activities and effects. By properly matching these extracts, the synergistic effects between them can be fully utilized to provide comprehensive anti-wrinkle and antioxidant effects for the skin.

[0017] It should be noted that ginseng root extract is usually prepared by extracting ginseng root by water extraction, but the inventors of the present application have found through a large number of experimental verifications that parameters such as the solid-to-liquid ratio and the number of water extractions of ginseng root extract have an important influence on the type and content of ginsenosides. When ginseng root extract is prepared by water extraction of ginseng root twice under a specific solid-to-liquid ratio, the saponin component and content in the ginseng root extract are more appropriate. The obtained ginseng root extract is combined with the remaining two ginseng-related components to obtain a ginseng composition with better antioxidant efficacy.

[0018] In a specific embodiment of the present invention, in the method for preparing the ginseng root extract, the time for the two water extractions is 1 to 3 hours.

[0019] Preferably, in the method for preparing the ginseng root extract, the water extraction further includes an ultrafiltration step, wherein the ultrafiltration includes collecting the filtrate using an ultrafiltration membrane with a molecular weight of 2000 to 5000 Da.

[0020] More preferably, in the preparation method of the ginseng root extract, the ultrafiltration further comprises a concentration step, wherein the concentration comprises concentrating the filtrate to a solid content of 30-40 wt %. It should be noted that solids will precipitate in the system due to evaporation of the extraction solvent during the concentration process.

[0021] In a specific embodiment of the present invention, in the method for preparing the ginseng root extract, the raw material is sieved using a 1.2 cm sieve after the pulverization.

[0022] In a specific embodiment of the present invention, in the method for preparing the ginseng root extract, the step of filtering with a ceramic membrane is further included before the ultrafiltration, and the pore size of the ceramic membrane is 200 nm.

[0023] In a specific embodiment of the present invention, in the method for preparing the ginseng root extract, the concentration is performed using an external circulation concentrator, and the concentration is performed at a temperature of 50 to 70°C.

[0024] In a specific embodiment of the present invention, in the preparation method of the ginseng root extract, the concentration further includes the steps of centrifugation, sterilization, drying and crushing, the centrifugation speed is 13000-15000 rpm, the sterilization is carried out at 115-135°C, the drying includes spray drying under the conditions of inlet air temperature of 185-210°C and outlet air temperature of 85-105°C, and the crushing is followed by passing through an 80-mesh sieve.

[0025] Preferably, the ginseng root extract comprises the following ginsenosides calculated by mass percentage:

[0026] 3~6wt% Rb1, 3~6wt% Rc, 3~4wt% Rb2, 0.1~0.5wt% Rb3, 1~2wt% Rd.

[0027] Preferably, the ginseng fruit extract includes the following prototype ginsenosides calculated by mass percentage:

[0028] 1~3wt%Rg1, 25~35wt%Re, 0.05~0.5wt%Rf, 15~21wt%Rd, 0.05~1wt%Rb1, 4~9wt%Rc, 4~9wt%Rb2, 1~3wt%Rb3.

[0029] More preferably, the ginseng fruit extract further comprises the following rare ginsenosides calculated by mass percentage:

[0030] 1~2wt% S-Rh1, 0.5~1wt% S-Rg2, 3~4.5wt% R-Rg2, 4~5.5wt% R-Rh1, 5~6wt% S-Rg3, 1~2wt% R-Rg3, 0.5 ~1.5wt%Rg5, 0.5~1wt%S-Rh2, 0.1~0.5wt%R-Rh2, 5~7wt%F4, 4~5wt%Rh4, 1~3wt%Rk1, 0.5~1wt%Rk3.

[0031] Preferably, the ginseng essential oil contains the following ginsenosides calculated by mass concentration:

[0032] 0.06~0.08mg / g Rb1, 0.06~0.08mg / g Rc, 0.06~0.09mg / g S-Rg3, 0.01~0.06mg / g R-Rg3, 0.05~0.2mg / g Rg5, 0.5~1.5mg / g Rk1.

[0033] The present invention also protects a method for preparing the ginseng composition, comprising the following steps:

[0034] The ginseng root extract, ginseng fruit extract, ginseng essential oil and water are mixed until no layers are separated to obtain a ginseng composition.

[0035] Preferably, the ginseng essential oil further comprises a pre-dissolution step before mixing with water, and the pre-dissolution comprises dissolving the ginseng essential oil with polyoxyethylene hydrogenated castor oil.

[0036] The present invention also protects the application of the ginseng composition in skin care products.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The ginseng composition provided by the present invention realizes the best use of ginseng. The scavenging rate of the composition for DPPH is as high as 55.14±1.42% or more, which is significantly better than a single component or a formula replaced by other similar components. It shows a significant ability to scavenge free radicals and excellent antioxidant potential. In the anti-wrinkle efficacy test, the inhibition rate of zebrafish tail fin wrinkling is as high as 53.17%, which has excellent anti-wrinkle efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The present invention provides a preparation process for the ginseng composition.

[0040] Figure 2 The HPLC chromatogram of ginsenosides in ginseng essential oil used in the present invention.

[0041] Figure 3 The figure is a result diagram of the scavenging rate of DPPH free radicals by the compositions obtained in the examples of the present invention and the comparative examples.

[0042] Figure 4 This is a test data chart of the anti-wrinkle efficacy of Example 1 and Comparative Examples 1 to 3 of the present invention on zebrafish.

[0043] Figure 5 This is a test diagram of the anti-wrinkle effect of Example 1 and Comparative Examples 1 to 3 of the present invention on zebrafish. DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the raw materials and reagents used in the embodiments of the present invention are conventionally purchased raw materials and reagents. Among them, the raw material information used in each embodiment and comparative example is as follows:

[0045] Ginseng fruit extract: purchased from Ji'an Hongsheng Biotechnology Co., Ltd.

[0046] Ginseng essential oil: purchased from Zhenji Ginseng Biotechnology (Changchun) Co., Ltd.

[0047] Lavender essential oil: purchased from Man's (Shanghai) Flavors & Fragrances Co., Ltd.

[0048] Macadamia nut seed oil: purchased from Laibaokang Daily Chemical (Shanghai) Co., Ltd.

[0049] Sea buckthorn fruit oil: purchased from Qinghai Kangpu Biotechnology Co., Ltd.

[0050] Briechera fruit oil: purchased from Shanghai Lishengshengte Enterprise Development Co., Ltd.

[0051] Gentiana root extract: purchased from Guangzhou Jianeng Biotechnology Co., Ltd.

[0052] Aloe vera extract: purchased from Yunnan Wanlv Biological Co., Ltd.

[0053] Polyoxyethylene hydrogenated castor oil RH-40: purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0054] 2,2-Biphenyl-1-picrylhydrazyl (DPPH reagent): purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0055] Ginsenoside Re: batch number: 110754-201827, purchased from China Food and Drug Inspection Institutes.

[0056] Ginsenoside Rg1: batch number: 110703-201731, purchased from China Food and Drug Inspection Institutes.

[0057] Ginsenoside Rd: batch number: 111818-201603, purchased from China Food and Drug Inspection Institutes.

[0058] Ginsenoside Rb3: Batch number: 111686-201504, purchased from China Food and Drug Inspection Institutes.

[0059] Ginsenoside Rb2: batch number: 111715-201203, purchased from China Food and Drug Inspection Institutes.

[0060] Ginsenoside S-F1: 20(S)-F1, batch number: 111763-201602, was purchased from China Food and Drug Inspection Institutes.

[0061] Ginsenoside S-F2: 20(S)-F2, batch number: 111764-201602, was purchased from China Food and Drug Inspection Institutes.

[0062] 20(S)-protopanaxatriol: PPT, batch number: 111764-201602, purchased from China Food and Drug Inspection Institutes.

[0063] Ginsenoside Rb1: batch number: 110704-201726, purchased from China Food and Drug Inspection Institutes.

[0064] Ginsenoside Rc: batch number: P23011L122926, purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0065] Ginsenoside S-Rg3: 20(S)-Rg3, batch number: A12GB144612, was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0066] Ginsenoside Rh4: Batch number: B24742-5mg, purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0067] Ginsenoside S-Rg2: 20(S)-Rg2, batch number: 63200010, was purchased from Shanghai Anpu Laboratory Technology Co., Ltd.

[0068] Ginsenoside Rf: Batch number: 65250020, purchased from Shanghai Anpu Laboratory Technology Co., Ltd.

[0069] Ginsenoside S-Rh1: 20(S)-Rh1, batch number: 2600010, was purchased from Shanghai Anpu Laboratory Technology Co., Ltd.

[0070] Ginsenoside R-Rh1: 20(R)-Rh1, batch number: B7910002, was purchased from Shanghai Anpu Laboratory Technology Co., Ltd.

[0071] Ginsenoside S-Rh2: 20(S)-Rh2, batch number: Y270010, was purchased from Shanghai Anpu Laboratory Technology Co., Ltd.

[0072] Ginsenoside R-Rh2: 20(R)-Rh2, batch number: ST05670120, was purchased from Shanghai Shidande Standard Technology Service Co., Ltd.

[0073] Ginsenoside CK: Batch number: ST11200120-9287, purchased from Shanghai Shidande Standard Technology Service Co., Ltd.

[0074] 20(S)-Protopanaxadiol: PPD, batch number: RS00991020-5550, was purchased from Shanghai Standard Standard Technology Service Co., Ltd.

[0075] Ginsenoside F4: batch number ST18950105-14186, purchased from Shanghai Shidande Standard Technology Service Co., Ltd.

[0076] Ginsenoside Rk1: Batch number: ST13360320-13012, purchased from Shanghai Shidande Standard Technology Service Co., Ltd.

[0077] Ginsenoside Rk3: Batch number: ST23220205-9131, purchased from Shanghai Shidande Standard Technology Service Co., Ltd.

[0078] Ginsenoside R-Rg3: 20(R)-Rg3, batch number: E-0648-98%-10 mg, purchased from Shanghai Tongtian Biotechnology Co., Ltd.

[0079] Ginsenoside Rg5: Shidande, ST21290310-10859, purity 93.7%.

[0080] Ginsenoside Rg6: Source: Leaf Biotechnology, A17IB223051, purity 98.3%.

[0081] Ginsenoside R-Rg2: HY-N2039 was purchased from Shanghai Tongtian Biotechnology Co., Ltd.

[0082] Ginseng root: including main root and lateral root, purchased from Liaoning Hengyuantang Pharmaceutical Co., Ltd.

[0083] Examples 1 to 4 and Comparative Examples 1 to 12

[0084] This embodiment and comparative example provide a series of ginseng compositions with different composition ratios, and the preparation method thereof comprises the following steps:

[0085] Polyoxyethylene hydrogenated castor oil is used to pre-dissolve ginseng essential oil, ginseng root extract is dissolved in water at room temperature to obtain a 10 mg / g ginseng root extract aqueous solution, and then ginseng fruit extract and ginseng essential oil are respectively dissolved in water at 80° C. to obtain a 10 mg / g ginseng fruit extract aqueous solution and a 10 mg / g ginseng essential oil solution, and the three are mixed and diluted according to a ratio to obtain a ginseng composition with a total concentration of 1.25 mg / g;

[0086] The preparation method of ginseng root extract comprises the following steps:

[0087] The ginseng root is weighed and crushed in a mass ratio of 1:2, and the raw material is sieved with a 1.2 cm sieve, and water extraction is performed twice at 90°C. The time of each water extraction is 2 hours, the solid-liquid ratio of the first water extraction is 1:10, and the solid-liquid ratio of the second water extraction is 1:8. After extraction, the filtrate is filtered and collected using a ceramic membrane (pore size of 200 nm) and an ultrafiltration membrane (3500 Da), and concentrated to a solid content of 35% at a temperature of 60°C through an external circulation concentrator. The obtained concentrated solution is centrifuged at 14000 rpm, the clear liquid is taken, sterilized at 125°C, spray-dried under the conditions of an inlet air temperature of 195°C and an outlet air temperature of 95°C, and then crushed and passed through an 80-mesh sieve to obtain a ginseng root extract.

[0088] The specific formulations of the ginseng compositions in Examples 1 to 4 and Comparative Examples 1 to 12 are shown in Table 1 below:

[0089] Table 1. Specific mass fractions of ginseng compositions in Examples 1 to 4 and Comparative Examples 1 to 12

[0090]

[0091] Comparative Example 13

[0092] A ginseng composition, which is different from Example 1 only in that:

[0093] Replace the ginseng essential oil with an equal amount of lavender essential oil.

[0094] Comparative Example 14

[0095] A ginseng composition, which is different from Example 1 only in that:

[0096] The ginseng fruit extract was replaced with an equal amount of macadamia seed extract.

[0097] Comparative Example 15

[0098] A ginseng composition, which is different from Example 1 only in that:

[0099] The ginseng fruit extract was replaced with an equal amount of sea buckthorn fruit extract.

[0100] Comparative Example 16

[0101] A ginseng composition, which is different from Example 1 only in that:

[0102] The ginseng fruit extract was replaced with an equal amount of bridge fruit extract.

[0103] Comparative Example 17

[0104] A ginseng composition, which is different from Example 1 only in that:

[0105] The ginseng root extract was replaced with an equal amount of gentian root extract.

[0106] Comparative Example 18

[0107] A ginseng composition, which is different from Example 1 only in that:

[0108] The ginseng root extract was replaced with an equal amount of aloe vera extract.

[0109] Comparative Example 19

[0110] A ginseng composition, which is different from Example 1 only in that:

[0111] In the preparation method of ginseng root extract, the water extraction operation is as follows:

[0112] Water extraction was carried out at 90℃ for 4h, with a solid-liquid ratio of 1:20.

[0113] Performance Testing

[0114] 1. Ginsenoside component detection

[0115] Next, the ginsenoside components of the ginseng root extract, ginseng fruit extract and ginseng essential oil in Example 1 were detected.

[0116] 1.1 Ginseng Root Extract

[0117] 1.1.1 Detection method:

[0118] S1. Prepare ginsenoside standard mixed solution: accurately weigh 7 mg of each of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rc and ginsenoside Rd standard products into a 25 ml volumetric flask, dissolve them by ultrasonication with about 20 ml of methanol, let them cool to room temperature, dilute to the mark with methanol, take 1 ml to dilute to the mark in a 10 ml volumetric flask, shake well, and obtain the standard solution;

[0119] S2. Sample treatment: Accurately weigh about 150 mg of ginseng root extract, put it into a 50 mL volumetric flask, add about 40 mL of methanol, and after ultrasonic vibration for about 30 minutes, let it cool to room temperature, dilute to the mark with methanol, and filter with a 0.45 μm needle filter;

[0120] S3. Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler (4.6×250 mm), acetonitrile was used as the mobile phase A, and 0.1% phosphoric acid solution was used as the mobile phase B. Gradient elution was performed according to the procedure in the following table:

[0121] Column temperature: 30°C, detection wavelength: 203 nm, flow rate: 1.0 mL / min;

[0122]

[0123] S4. The ginseng root extract is passed into a high performance liquid chromatograph for detection.

[0124] 1.1.2 Test results

[0125] The components and contents of ginsenosides in ginseng root extract are shown in Table 2 below:

[0126] Table 2. Ginsenoside components and contents in ginseng root extract

[0127]

[0128]

[0129] 1.2. Ginseng fruit extract

[0130] 1.2.1 Detection method:

[0131] S1. Preparation of test sample: accurately weigh about 0.1 g of ginseng fruit extract sample, place in a 25.00 mL volumetric flask, add about 20 mL of methanol and shake gently, extract by ultrasonic (45 kHz, 290 W) for 20 min, cool to room temperature, dilute to 25.00 mL, shake well, filter the supernatant with a 0.22 μm filter membrane, and take the filtrate;

[0132] S2. Preparation of reference solution: accurately weigh 20(S)-Rg2, 20(R)-Rg2, 20(S)-Rg3, 20(R)-Rg3, Rd, Re, Rf, Rg1, Rg5, 20(S)-Rh1, 20(R)-Rh1, Rb1, Rb2, Rb3, Rc, 20(S)-Rh2, 20(R)-Rh2, 20(S)-F1, 20(S)-F2, CK, PPD, PPT, F4, Rh4, Rk1, Rk3 ginsenosides, respectively. methanol and dilute to the mark, shake well to obtain 4.214, 3.943, 4.991, 1.86, 4.633, 4.005, 1.899, 3.709, 3.642, 3.784, 3.992, 3.680, 1.995, 0.169, 1.38, 1.92, 3.714, 1.881, 1.877, 2.034, 1.888, 1.850, 0.382, 0.355, 1.081, 0.376 mg / mL reference solution;

[0133] S3. The UPLC method was used to detect the contents of 22 ginsenosides, including Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1, S-Rg2, R-Rg2, S-Rg3, R-Rg3, Rg5, S-Rh1, R-Rh1, S-Rh2, R-Rh2, F1, F2, CK, PPD and PPT. The chromatographic column used was Acquity BEH Shield RP18 (2.1 mm × 100 mm, 1.7 μm), and Acquity CSH C18 (2.1mm×100mm, 1.7μm), detection wavelength 203nm; injection volume 2μL; column temperature 40℃; mobile phase acetonitrile (A)-water (B), flow rate 0.6mL / min, gradient elution program see the table below:

[0134]

[0135]

[0136] S4. The contents of F4, Rh4, Rk1 and Rk3 were detected by HPLC. The chromatographic column was Hungpu T3 (4.6 mm × 250 mm, 5 μm), the column temperature was 35 °C, the injection volume was 20 μL, the detection wavelength was 203 nm, the mobile phase was acetonitrile (A)-water (B), and the flow rate was 1.0 mL / min. The gradient elution program is shown in the table:

[0137]

[0138] 1.2.2 Test results

[0139] The contents of ginsenosides in ginseng fruit extract are shown in Table 3, among which ginsenosides Rb1, Rc, Rb2, Rb3, Rg1, Re, Rf, and Rd are prototype ginsenosides, and the rest are rare ginsenosides:

[0140] Table 3. Content of ginsenosides in ginseng fruit extract

[0141]

[0142] 1.3. Ginseng essential oil

[0143] 1.3.1 Detection method:

[0144] S1. Prepare reference solutions of 8 saponins (Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1): accurately weigh about 10 mg of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, and ginsenoside Rg1 respectively and place them in the same 20 mL volumetric flask, add an appropriate amount of methanol to dissolve, and continue to add methanol to the scale line to obtain a mixed reference solution with a concentration of each ginsenoside of about 0.5 mg / mL;

[0145] S2. Prepare reference solutions of three saponins (S-Rg3, R-Rg3, Rg5): accurately weigh about 10 mg of 20(S)-ginsenoside Rg3, 20(R)-ginsenoside Rg3, and ginsenoside Rg5 reference substances respectively and place them in the same 50 mL volumetric flask, add an appropriate amount of methanol to dissolve, and continue to add methanol to the scale line to obtain a mixed reference solution with a concentration of each ginsenoside of about 0.2 mg / mL;

[0146] S3. Prepare reference solutions of four saponins (Rh4, Rk1, Rk3, F4): accurately weigh about 10 mg of ginsenoside Rh4, ginsenoside Rk1, ginsenoside Rk3, and ginsenoside F4 reference substances respectively and place them in the same 25 mL volumetric flask, add an appropriate amount of methanol to dissolve, and continue to add methanol to the scale line to obtain a mixed reference solution with a concentration of each ginsenoside of about 0.4 mg / mL;

[0147] S4. Prepare the reference solution of Rg6: accurately weigh about 10 mg of ginsenoside Rg6 reference substance into a 25 mL volumetric flask, add an appropriate amount of methanol to dissolve, and continue to add methanol to the scale line to obtain a ginsenoside Rg6 reference solution with a concentration of about 0.4 mg / mL;

[0148] S5. Prepare the test solution: accurately weigh about 1.0 g of ginseng essential oil sample and place it in a 10 mL volumetric flask, add an appropriate amount of methanol, ultrasonically (45 kHz 380 W) for 30 minutes, remove and cool, continue to add methanol to the scale line, shake well, filter through a 0.22 μm microporous membrane, and take the filtrate to obtain;

[0149] S6. Detection of the contents of 8 saponins (Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1):

[0150] The HPLC method was used and the chromatographic conditions were as follows:

[0151] Chromatographic column: ACE Excel 5C18-AR (4.6 mm × 250 mm, 5 μm);

[0152] Mobile phase: (A) acetonitrile; (B) water;

[0153] Flow rate: 1.0 mL / min;

[0154] Detection wavelength: 203nm;

[0155] Injection volume: 10 μL;

[0156] Column temperature: 35°C;

[0157] The gradient elution program is as follows:

[0158]

[0159]

[0160] S7. Detection of the contents of three saponins (S-Rg3, R-Rg3, Rg5):

[0161] The HPLC method was used, and the chromatographic conditions were consistent with those of S6, with the only difference being the gradient elution procedure as shown in the following table:

[0162]

[0163] S8. Detection of the contents of 5 saponins (Rh4, Rk1, Rk3, F4, Rg6):

[0164] The HPLC method was used. The chromatographic conditions were consistent with those of S6, with the only difference being that the chromatographic column was Hungpu T3 (4.6 mm × 250 mm, 5 μm), the injection volume was 20 μL, and the gradient elution program was as follows:

[0165]

[0166]

[0167] 1.3.2 Test results

[0168] The test was repeated twice, and the ginsenoside components and contents in ginseng essential oil were shown in Table 4 below:

[0169] Table 4. Ginsenoside components and contents in ginseng essential oil

[0170]

[0171] The HPLC chromatogram of ginsenosides in ginseng essential oil is as follows: Figure 2 shown.

[0172] 2. DPPH clearance test of ginseng composition

[0173] 2.1 Test Method

[0174] Prepare 0.2mmol / L DPPH solution with anhydrous ethanol, take 2mL DPPH solution and 2mL sample solution of different mass concentrations and mix them, react at room temperature in the dark for 30min, measure the absorbance at 517nm (A1), then replace the DPPH solution with 2mL anhydrous ethanol, measure the absorbance at 517nm (A2), finally replace the sample solution with 2mL distilled water, measure the absorbance at 517nm (A0).

[0175] The above experiment was performed in parallel three times. 200 μL of the solution was taken into a 96-well plate using a pipette, and the absorbance at 517 nm was measured using an ELISA reader.

[0176] The calculation formula of DPPH free radical scavenging rate (R1,%) is as follows:

[0177]

[0178] 2.2 DPPH clearance test operation and results of single components in ginseng composition

[0179] Prepare vitamin C positive control solution: accurately weigh an appropriate amount of ascorbic acid sample, add an appropriate amount of pure water, and prepare 2 mg / g, 1 mg / g, 0.5 mg / g, 0.25 mg / g, and 0.125 mg / g vitamin C solutions.

[0180] Preparation of ginseng root extract solution: Weigh appropriate amounts of ginseng root extract and dissolve them in pure water to prepare 10 mg / g, 5 mg / g, 2.5 mg / g, 1.25 mg / g, and 0.625 mg / g ginseng root extract solutions.

[0181] Preparation of ginseng fruit extract solution: Weigh appropriate amounts of ginseng fruit extract, dissolve in pure water, and heat to 80°C to dissolve to obtain 10 mg / g, 5 mg / g, 2.5 mg / g, 1.25 mg / g, and 0.625 mg / g ginseng fruit extract solutions.

[0182] Preparation of ginseng essential oil solution: weigh ginseng essential oil and RH-40 in a mass ratio of 1:2, heat and stir at 80°C to dissolve, add purified water to prepare 10 mg / g, 5 mg / g, 2.5 mg / g, 1.25 mg / g, and 0.625 mg / g ginseng essential oil solutions.

[0183] The DPPH scavenging rate of a single component in the ginseng composition at different concentrations (10 mg / g, 5 mg / g, 2.5 mg / g, 1.25 mg / g, 0.625 mg / g) was tested. The results are shown in Table 5:

[0184] Table 5. Scavenging rate of DPPH by a single component in the ginseng composition at different concentrations

[0185]

[0186]

[0187] According to the data in Table 5, the IC 50 The concentration is between 1.25mg / g and 2.5mg / g, ginseng essential oil IC 50 The concentration is between 5 mg / g and 10 mg / g. Therefore, in order to facilitate the observation of the synergistic effect of the composition in the later stage, the mass concentration of 1.25 mg / g is selected as the fixed value of the total concentration of the composition in the present invention.

[0188] 2.3 DPPH clearance test results of ginseng composition

[0189] The scavenging rate of DPPH by the ginseng composition obtained in the examples and comparative examples was tested according to the test method in section 2.1. The results are shown in Table 6 and Table 6 below. Figure 3 As shown:

[0190] Table 6. DPPH removal rate of ginseng compositions obtained in Examples and Comparative Examples

[0191]

[0192]

[0193] According to Table 6 and Figure 3 It can be seen that at the same mass concentration (each component is 1.25 mg / g), the DPPH scavenging rate of the ginseng composition obtained in the example is as high as 55.14±1.42% or more, which is significantly higher than that of the comparative example, indicating that the ginseng composition has a synergistic effect.

[0194] According to Comparative Examples 1 to 12, the ginseng composition of the present invention only includes any one or two of ginseng root extract, ginseng fruit extract, and ginseng essential oil, or an improper ratio, which will lead to a decrease in the synergistic effect and the DPPH clearance rate is not as good as the ginseng composition in the embodiment of the present invention.

[0195] According to Comparative Examples 13 to 18, different essential oils (lavender essential oil), fruit extracts (macadamia nut seed extract, sea buckthorn fruit extract, bridge fruit extract), and root extracts (gentian root extract, aloe vera extract) were used to replace ginseng essential oil, ginseng fruit extract, and ginseng root extract, respectively, and the antioxidant effects of the obtained compositions were not as good as the ginseng composition of the present invention. Comparative Example 19 used ginseng root extracts made from different processes to replace the original ginseng root extract. The DPPH scavenging rate was 49.28%, and its antioxidant effect was not as high as the scavenging rate of 57.08% in the corresponding Example 1.

[0196] 2.4 Significance Test

[0197] The DPPH removal rates of the compositions obtained in the examples and comparative examples were tested for significance, and the results are shown in Table 7 below:

[0198] Table 7. Significance between Examples and Comparative Examples

[0199]

[0200]

[0201] Note: P<0.05 was significant, P<0.01 was very significant.

[0202] According to Table 7 above, there are significant differences between the results of the embodiment and the comparative example, indicating that the scavenging effect of the ginseng composition obtained in the embodiment of the present invention on DPPH is significantly better than that of the comparative example.

[0203] 3. Anti-wrinkle efficacy test and results of ginseng combination

[0204] The structure of zebrafish skin is similar to that of human skin, so it can be used as a model to evaluate the anti-wrinkle effect of samples. UV irradiation will cause changes in collagen and elastin in the skin structure, causing the fish fins to shrink and deform. The anti-wrinkle effect of the test substance is evaluated by evaluating the degree to which the test substance slows down the shrinkage of the zebrafish tail fin.

[0205] The test method for zebrafish anti-wrinkle efficacy is as follows:

[0206] S1. Add embryo culture medium to the normal control group according to the well plate settings, add test substance solutions of different concentrations to each well of the test group, add benzophenone solution to the positive control group, cover the culture plate panel, protect from light, and incubate in a biochemical incubator at 28.5±0.5℃ for 2h;

[0207] S2. Use ultraviolet light therapy to irradiate the model group, positive control group and test group. After irradiation, wrap the culture plate with aluminum foil and incubate it in a constant temperature incubator at 28.5±0.5℃ in the dark for 22h;

[0208] S3. Observation and photography: 10 zebrafish were randomly selected from each group for observation and photography under a stereo microscope, and the images were saved for statistical analysis of the tail fin area of ​​each group of zebrafish;

[0209] S4. Image analysis: Use graphic analysis software to count the area of ​​the zebrafish caudal fin from the cloaca to the end of the caudal fin, calculate the mean and standard deviation of each group of experiments, and calculate the inhibition rate of caudal fin wrinkling;

[0210]

[0211] S5. Data processing: Statistical analysis software was used to perform variance analysis on the data, with the model group as the standard. P<0.05 indicated a significant difference. The tail fin wrinkle inhibition rate was positive and statistically different from that of the model group (P<0.05), indicating that the test substance had anti-wrinkle effects.

[0212] The compositions obtained in Example 1 and Comparative Examples 1 to 3 were used for anti-wrinkle test. The results are shown in Table 8 and Figures 4-5 As shown:

[0213] Table 8. Anti-wrinkle efficacy test results

[0214]

[0215] Note: Compared with the model group, "*" indicates P﹤0.05, "**" indicates P﹤0.01, "***" indicates P﹤0.001, "****" indicates P﹤0.0001, and "ns" indicates no significant difference.

[0216] From the above table 8 and Figures 4-5 It can be seen that at a concentration of 3.125 μg / ml, the tail fin area of ​​zebrafish in Example 1 was significantly increased compared with the model group (P<0.01), having an anti-wrinkle effect, with a very significant difference; Comparative Examples 1, 2 and 3 did not have anti-wrinkle effects.

[0217] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A ginseng composition with anti-wrinkle effect, characterized in that: It includes the following components calculated by mass: 1-2 parts of ginseng root extract, 1-2 parts of ginseng fruit extract, 1-2 parts of ginseng essential oil; The preparation method of the ginseng root extract comprises the following steps: Ginseng roots and rhizomes are crushed in a mass ratio of 1:(1-3), and water-extracted twice at 90-100°C, with the solid-liquid ratio of the first water extraction being 1:(9-11) and the solid-liquid ratio of the second water extraction being 1:(7-9).

2. The ginseng composition with anti-wrinkle effect as claimed in claim 1, characterized in that: In the preparation method of the ginseng root extract, the step of ultrafiltration is further included after the water extraction, and the ultrafiltration includes collecting the filtrate using an ultrafiltration membrane with a molecular weight of 2000-5000Da.

3. The ginseng composition with anti-wrinkle effect as claimed in claim 2, characterized in that: In the preparation method of the ginseng root extract, the ultrafiltration further includes a concentration step, and the concentration includes concentrating the filtrate to a solid content of 30-40wt%.

4. The ginseng composition with anti-wrinkle effect as claimed in claim 1, characterized in that: The ginseng root extract includes the following ginsenosides calculated by mass percentage: 3~6wt% Rb1, 3~6wt% Rc, 3~4wt% Rb2, 0.1~0.5wt% Rb3, 1~2wt% Rd.

5. The ginseng composition with anti-wrinkle effect as claimed in claim 1, characterized in that: The ginseng fruit extract includes the following prototype ginsenosides calculated by mass percentage: 1~3wt%Rg1, 25~35wt%Re, 0.05~0.5wt%Rf, 15~21wt%Rd, 0.05~1wt%Rb1, 4~9wt%Rc, 4~9wt%Rb2, 1~3wt%Rb3.

6. The ginseng composition with anti-wrinkle effect as claimed in claim 5, characterized in that: The ginseng fruit extract also includes the following rare ginsenosides calculated by mass percentage: 1~2wt% S-Rh1, 0.5~1wt% S-Rg2, 3~4.5wt% R-Rg2, 4~5.5wt% R-Rh1, 5~6wt% S-Rg3, 1~2wt% R-Rg3, 0.5 ~1.5wt%Rg5, 0.5~1wt%S-Rh2, 0.1~0.5wt%R-Rh2, 5~7wt%F4, 4~5wt%Rh4, 1~3wt%Rk1, 0.5~1wt%Rk3.

7. The ginseng composition with anti-wrinkle effect as claimed in claim 1, characterized in that: The ginseng essential oil contains the following ginsenosides calculated by mass concentration: 0.06~0.08mg / g Rb1, 0.06~0.08mg / g Rc, 0.06~0.09mg / g S-Rg3, 0.01~0.06mg / gR-Rg3, 0.05~0.2mg / g Rg5, 0.5~1.5mg / g Rk1.

8. The method for preparing the ginseng composition with anti-wrinkle effect according to any one of claims 1 to 7, characterized in that: The steps include: The ginseng root extract, ginseng fruit extract, ginseng essential oil and water are mixed until no layers are separated to obtain a ginseng composition.

9. The method for preparing the ginseng composition with anti-wrinkle effect as claimed in claim 8, characterized in that: The ginseng essential oil also includes a pre-dissolution step before mixing with water, and the pre-dissolution includes using polyoxyethylene hydrogenated castor oil to dissolve the ginseng essential oil.

10. Use of the ginseng composition with anti-wrinkle effect according to any one of claims 1 to 7 in skin care products.

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