A method for the biosynergistic extraction and refined utilization of all active ingredients in ginseng

Abstract

This invention proposes a method for the biosynergistic extraction and refined utilization of all active ingredients in ginseng, belonging to the fields of medical, dental, or cosmetic formulations. This invention utilizes ginseng stress extract obtained through specific microbial treatment. This method leverages the stress stimulation of *Pseudomonas fluorescens* to efficiently synthesize various saponins, polysaccharides, and volatile oils in ginseng, resulting in an extract rich in components and with enhanced activity. Combining fractional extraction, resin purification, and nanoemulsification processes, a skincare raw material with comprehensive components, high stability, and excellent bioavailability is ultimately obtained, exhibiting a powerful synergistic effect in anti-inflammatory and anti-aging properties.

Description

Technical Field

[0001] This invention belongs to the field of medical, dental or cosmetic formulations, and particularly relates to a method for the biosynergistic extraction and refined utilization of all active ingredients of ginseng. Background Technology

[0002] Ginseng (Panax ginseng CA Mey.), a perennial herb belonging to the genus Panax in the family Araliaceae, is not only a highly representative and precious traditional Chinese medicine, but also a popular functional ingredient in modern medicine, health products, and cosmetics. Its medicinal history can be traced back more than two thousand years. The *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) records it as "primarily nourishing the five internal organs, calming the spirit, stabilizing the soul, stopping palpitations, dispelling evil influences, brightening the eyes, opening the heart and improving intelligence, and long-term use lightening the body and prolonging life," establishing its status as the "King of Herbs." With the development of modern science and technology, research on ginseng has deepened, achieving significant results in areas ranging from its botanical characteristics and the isolation of its active ingredients to the optimization of extraction techniques and the expansion of its applications in multiple fields.

[0003] The efficacy of ginseng is closely related to its complex chemical composition. After decades of research, researchers have isolated and identified a variety of bioactive chemical components from ginseng. Among them, ginsenosides, ginseng polysaccharides, ginseng polypeptides, volatile oils, amino acids and trace elements are the main active ingredients. These components work together to give ginseng a variety of physiological activities.

[0004] (a) Ginsenosides

[0005] Ginsenosides are the most representative active ingredients in ginseng and are also the most extensively studied class of compounds, accounting for approximately 2%-6% of the dry weight of ginseng. To date, more than 100 ginsenoside monomers have been isolated from ginseng. Based on the differences in the chemical structure of the sapogenins, they can be divided into three main categories: diol-type ginsenosides (represented by Rb1, Rb2, Rc, Rd, etc.), triol-type ginsenosides (represented by Rg1, Rg2, Re, Rh1, etc.), and oleanolic acid-type ginsenosides (represented by Ro). Different types of ginsenosides possess different physiological activities. For example, Rb1, a diol-type ginsenoside, exhibits significant anti-fatigue and neuroprotective effects, improves learning and memory, and regulates central nervous system function. Rg1, a triol-type ginsenoside, is particularly effective in promoting protein synthesis, enhancing immunity, and improving cardiovascular circulation, while also promoting bone marrow hematopoiesis. Oleanolic acid-type ginsenoside Ro has anti-inflammatory, hepatoprotective, and lipid-lowering effects. Furthermore, ginsenosides undergo transformation during processing. For instance, during the steaming process of red ginseng, some diol-type ginsenosides are converted into rare ginsenosides such as Rg3 and Rh2. These rare ginsenosides possess stronger anti-tumor and antioxidant activities, further enhancing the medicinal value of ginseng.

[0006] (ii) Ginseng polysaccharides

[0007] Ginseng polysaccharides are another important active ingredient in ginseng, accounting for approximately 10%-20% of the dry weight of ginseng. They are mainly composed of monosaccharides such as glucose, galactose, arabinose, and rhamnose, and can be classified according to their origin as ginseng root polysaccharides, ginseng stem and leaf polysaccharides, and ginseng fruit polysaccharides. Ginseng polysaccharides have good water solubility and extremely low toxicity. They are excellent immunomodulators, significantly enhancing the body's immune function, promoting lymphocyte proliferation, macrophage phagocytic activity, and the secretion of cytokines (such as interleukin-2 and interferon-γ), thereby improving the body's resistance to pathogens. In addition, ginseng polysaccharides also have antioxidant, anti-aging, hypoglycemic, and hepatoprotective effects. Their antioxidant mechanism mainly involves scavenging free radicals in the body, inhibiting lipid peroxidation, and reducing oxidative stress damage to cells. In terms of hypoglycemia, ginseng polysaccharides can improve insulin sensitivity, promote liver glycogen synthesis, and inhibit liver glycogenolysis, thereby stabilizing blood sugar levels.

[0008] (iii) Other active ingredients

[0009] Besides ginsenosides and polysaccharides, ginseng also contains a variety of other bioactive minor components. Ginseng peptides are small molecule peptides composed of amino acids, characterized by easy absorption and high bioavailability. They can promote collagen synthesis in the skin, enhance skin elasticity, and also have certain antioxidant and immunomodulatory effects. Ginseng volatile oil is present in relatively low amounts, accounting for approximately 0.05%-0.2% of the dry weight of ginseng. Its main components are sesquiterpenes, alcohols, aldehydes, and other compounds, which have sedative, calming, and antibacterial effects. Its unique aroma also makes it suitable for both functional and fragrance purposes in cosmetics. In addition, ginseng contains more than 17 kinds of amino acids (including the 8 essential amino acids for the human body), more than 20 kinds of trace elements (such as potassium, sodium, calcium, magnesium, iron, zinc, selenium, etc.), and nutrients such as B vitamins and vitamin C. These components can provide nutritional support to the body, participate in cell metabolism, and help enhance the overall efficacy of ginseng.

[0010] The application of ginseng extract in skincare products is mainly based on its various skin health benefits:

[0011] Antioxidant effects: The saponins and polysaccharides in ginseng have significant antioxidant activity, which can scavenge free radicals such as DPPH, OH, and ABTS+, and reduce the damage of oxidative stress to the skin.

[0012] Anti-inflammatory effects: Ginseng and its active ingredients (such as ginsenosides Rb1, Rg3, Compound K, etc.) inhibit inflammatory responses such as atopic dermatitis and skin allergies by regulating inflammatory pathways such as NF-κB, MAPK, and PI3K / AKT.

[0013] Skin whitening and fading dark spots: Ginseng extract can inhibit tyrosinase activity, reduce melanin production, and at the same time reduce pigmentation through its antioxidant effect.

[0014] Moisturizing and hydrating: Ginseng polysaccharides have excellent moisturizing properties, which can enhance the moisture content of the stratum corneum and prevent dry skin.

[0015] Anti-aging: Ginseng can promote the proliferation of skin fibroblasts and collagen synthesis, improve skin elasticity, and reduce wrinkle formation.

[0016] Traditional extraction methods for ginseng's active ingredients mainly include water extraction and alcohol extraction. Water extraction typically involves slicing or crushing ginseng and then refluxing it with water. One method for extracting fresh ginseng involves placing the whole fresh ginseng root in purified water at 75-85℃ and refluxing for 3.5-4.5 hours. The resulting extract contains ginsenosides at a concentration of ≥40 mg / 100 ml. This method best preserves the pure taste and active ingredients of fresh ginseng.

[0017] Alcohol extraction uses organic solvents such as ethanol for extraction, and this method has a high extraction efficiency for active ingredients such as saponins. Studies have shown that ethanol concentration has a significant impact on extraction efficiency; extraction with 30% ethanol concentration and high-speed homogenization for 6 minutes can obtain a good extraction rate of total ginsenosides and polysaccharides.

[0018] With the development of science and technology, some modern extraction techniques have been applied to the extraction of active ingredients from ginseng to improve extraction efficiency and maintain ingredient activity. Enzymatic hydrolysis-assisted extraction is one such method. It utilizes enzymes such as pectinase to disrupt the cell wall structure of ginseng, thereby releasing the active ingredients more fully. Studies have shown that ginseng extracts obtained using enzymatic hydrolysis combined with high-speed homogenization can identify nine ginsenosides (Re, Rf, Rb1, Rc, Rb2, Rg2, Rd, Rg8, and Rg3), three more than those obtained using traditional extraction methods (Rd, Rg8, and Rg3).

[0019] High-speed homogenization is another highly efficient extraction technique. It breaks down plant cell walls through high-speed shearing and agitation, promoting the release of contents. This method offers short extraction times, high efficiency, and produces extracts with stronger antioxidant activity.

[0020] Fermentation is an emerging extraction technology that uses microbial fermentation to transform the large molecular components in ginseng into smaller, more easily absorbed active substances. For example, ginseng fermented with lactic acid bacteria can produce rare ginsenosides such as Rh2 and CK, which have enhanced activity and provide better moisturizing, whitening, antibacterial, and anti-aging effects in cosmetics.

[0021] In the extraction process of ginseng's active ingredients, traditional methods typically target the separation and purification of single-category active ingredients (such as polysaccharides, saponins, or oligopeptides), making it difficult to achieve efficient synergistic extraction of multiple components. In recent years, with continuous innovation in extraction technology, some novel extraction processes have been developed, aiming to simultaneously obtain multiple high-value active ingredients and enhance their bioactivity and application value.

[0022] CN118147260B discloses a method for co-extracting ginseng polypeptides and rare saponins. This method involves ginseng pulverization and crude extraction, protease digestion, rare saponin conversion, and multi-stage nanofiltration. It achieves the synergistic extraction of ginseng polypeptides and rare saponins, avoiding the destruction of active ingredients caused by multiple processing steps. The conversion rate of rare saponins is significantly improved, with Rg3 and Rh2 contents reaching 15%–16% and 5%–6% of the total saponins, respectively. The extract exhibits synergistic effects in anti-fatigue and immune-enhancing properties, making it suitable for the development of functional foods and pharmaceuticals.

[0023] CN119818549B discloses a method for preparing ginseng extract that does not cause internal heat: the steps include natural fermentation and ultrasonic treatment, compound enzymatic hydrolysis, enzyme inactivation and purification; the innovation of this method is that: the fermentation-ultrasonic-enzymatic hydrolysis triple process significantly reduces the content of components that cause internal heat, such as ginsenosides Rg1 and Rb1, which are not detected in the final product; the extract has a wider range of applications, especially suitable for people with a hot constitution or who are prone to internal heat; the process conditions are mild and can retain other active ingredients in ginseng (such as rare saponins and polysaccharides).

[0024] For ginseng, one of the core research directions is how to more effectively increase the content of its active ingredients or improve its extraction rate. Summary of the Invention

[0025] The purpose of this invention is to propose a method for the biosynergistic extraction and refined utilization of all active ingredients in ginseng, thereby increasing the content of its active ingredients through special means.

[0026] A ginseng stress extract, the preparation of which includes the following steps:

[0027] S1 Pretreatment: Fresh ginseng is washed and then soaked in stress solution for treatment;

[0028] The stress fluid contains the following components:

[0029] The activated fluorescent Pseudomonas suspension has a concentration ≥10 6 CFU / mL;

[0030] S2 induction culture: induction culture of pretreated fresh ginseng;

[0031] Induction conditions are: >90% RH, 20-25℃; protected from light;

[0032] S3 Sterilization and Separation: The ginseng after induction culture is sterilized and then separated into two parts: ginseng leaves and ginseng roots;

[0033] S4 Ginseng Leaf Extract: Ginseng leaves are dried, and then ginseng polypeptides, ginseng polysaccharides and ginsenosides are extracted and purified separately for later use;

[0034] S5 Ginseng Root Extract: Ginseng roots are dried and extracted and purified in the following order: volatile oil, ginseng polypeptides, ginsenosides, and ginseng polysaccharides; for later use.

[0035] S6 Mixing: Mix the components extracted in steps S4 and S5; this is the ginseng stress extract.

[0036] Preferably, in step S1, the stress solution further contains methyl o-hydroxybenzoate at a concentration ≥500 μM.

[0037] Preferably, the induction culture time in step S2 is at least 3 days.

[0038] Preferably, step S4, ginseng leaf extraction, involves: drying the ginseng leaves and passing them through a sieve of at least 40 mesh; first, extracting with pure water to separate the supernatant and residue; then extracting the residue with an ethanol-water solution; finally, concentrating the extract and eluting the concentrated solution with resin to remove ginseng polysaccharides and ginsenosides.

[0039] Preferably, the residue is extracted using ultrasound-assisted extraction, the ethanol-water solution has an ethanol concentration of at least 50 wt%, the material-to-liquid ratio during extraction is 1:≥15 g / mL, the extraction is performed by reflux at 40-50℃, and the extraction is performed at least twice, each time for at least 20 minutes.

[0040] When the concentrate is made of resin, ginseng polysaccharides are first eluted with pure water; then ginsenosides are eluted with an ethanol solution of ≥70% concentration.

[0041] The supernatant was concentrated under reduced pressure and precipitated with alcohol to obtain ginseng polysaccharide and ginseng polypeptide solutions. The ginseng polypeptide solutions were purified by chromatographic separation and then dried.

[0042] After elution, ginseng polysaccharides were concentrated, deproteinized, precipitated with alcohol, washed, and dried.

[0043] Ginsenosides were concentrated and dried after elution.

[0044] Preferably, step S5, ginseng root extraction, involves drying the ginseng root and passing it through a sieve of at least 40 mesh, then processing the ginseng root powder using steam distillation to obtain volatile oil and ginseng residue.

[0045] Water was added to the ginseng residue and enzymatic hydrolysis was performed using protease; after enzymatic hydrolysis, solid-liquid separation was performed to obtain a polypeptide aqueous extract and secondary residue;

[0046] The secondary residue was extracted with an ethanol-water solution; after extraction, the extract was concentrated, and the concentrate was eluted with resin to remove ginseng polysaccharides and ginsenosides.

[0047] Preferably, when using an ethanol-water solution for secondary residue extraction, ultrasonic-assisted extraction is performed, wherein the ethanol concentration in the ethanol-water solution is at least 50 wt%; the material-to-liquid ratio during extraction is 1:≥15 g / mL; reflux extraction is performed at 40-50℃; and the extraction is performed at least twice, each time for at least 20 min.

[0048] When the concentrate is made of resin, ginseng polysaccharides are first eluted with pure water; then ginsenosides are eluted with an ethanol solution of ≥70% concentration.

[0049] The aqueous extract of the polypeptide was concentrated under reduced pressure, filtered through an ultrafiltration membrane to obtain the filtrate, and the filtrate was precipitated with alcohol to obtain ginseng polysaccharide and ginseng polypeptide solution. The ginseng polypeptide solution was purified by chromatographic separation and then dried.

[0050] After elution, ginseng polysaccharides were concentrated, deproteinized, precipitated with alcohol, washed, and dried.

[0051] Ginsenosides were concentrated and dried after elution.

[0052] The second objective of this invention is to provide the application of the aforementioned ginseng stress extract in the preparation of cosmetics; primarily used as an anti-inflammatory and anti-aging ingredient.

[0053] Ginseng Stress Extract: Through stress response, ginseng's own defense mechanisms are activated, synthesizing and accumulating a wider variety of active substances such as ginseng polypeptides, ginsenosides, ginseng polysaccharides, and volatile oils. These components possess multiple effects, including moisturizing, repairing, anti-oxidation, and soothing, exerting anti-inflammatory and anti-aging effects through multiple pathways. Since the ginseng stress extract of this invention consists of both volatile oils and water-soluble components, it is generally prepared into a microemulsion using emulsifiers (such as lecithin) through high-pressure homogenization or other methods for use or storage; however, in special cases, separate packaging may also be used.

[0054] This invention employs bio-stress technology to treat fresh ginseng, using *Pseudomonas fluorescens* as an inducer to simulate the stress state of ginseng when it encounters pathogens in nature. This method is safer and more natural than chemical or physical stress methods (such as UV radiation or heavy metals), and produces more complex and abundant secondary metabolites, aligning with the trend of "natural" cosmetics.

[0055] This invention processes ginseng leaves and ginseng roots separately, adhering to the principle of "applying the right material to the right application." The components of ginseng leaves and roots differ; extracting them separately maximizes the retention of their respective active ingredients, avoids cross-interference, and improves extraction efficiency.

[0056] This invention ultimately produces a nanoemulsion from the extract. Nanotechnology significantly improves the solubility, stability, and bioavailability of the active ingredients, making them easier to penetrate the stratum corneum and exert their effects, which is crucial for skincare applications.

[0057] More specifically, this invention adds methyl salicylate (or methyl hydroxybenzoate) to the stress response; this is an important signaling molecule in plant disease resistance. When used in combination with *Pseudomonas fluorescens*, it can create a dual stress stimulus on ginseng, more strongly activating the ginseng's defense system, thereby significantly increasing the accumulation of target active substances such as ginsenosides and polysaccharides, surpassing the effect of single-microorganism induction.

[0058] The cultivation and adjustment period after stress are limited, as plant stress responses and the accumulation of secondary metabolites require time. This approach ensures sufficient time for the production of abundant active ingredients without excessively long production cycles, making industrial-scale production feasible.

[0059] Aside from differences in the content and specific composition of saponins and polysaccharides, the volatile oil components of ginseng roots and leaves are mainly concentrated in the roots. Therefore, they need to be processed separately. First, the volatile oil of ginseng roots is extracted, and then the specific extraction and purification process is defined to ensure that high-purity saponins and polysaccharides can be obtained.

[0060] This approach significantly enhances product efficacy through bio-stress technology. By utilizing *Pseudomonas fluorescens* to stimulate ginseng to efficiently synthesize various saponins, polysaccharides, and volatile oils, a richer and more active extract is obtained. Ultimately, a skincare ingredient with comprehensive components, high stability, and excellent bioavailability is produced, exhibiting a powerful synergistic effect in anti-inflammatory and anti-aging properties. Detailed Implementation

[0061] To better understand the present invention, the present invention will be further described below with reference to specific serial numbers. The terminology used in the serial numbers is for describing specific embodiments and does not constitute a limitation on the scope of protection of the present invention.

[0062] In the specific implementation methods, unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used are all commercially available unless otherwise specified.

[0063] Unless otherwise specified, percentages, % and so on in the specific implementation method are assumed to be mass percentages.

[0064] The raw materials used in this invention are as follows:

[0065] Fluorescent Pseudomonas: Pseudomonas fluorescens (CGMCC 1.823)

[0066] Bacillus subtilis: Bacillus subtilis Bacullus sbutilis (CGMCC No. 15250)

[0067] Trichoderma atroviride (CGMCC No. 17979)

[0068] Ginseng: Fresh ginseng (Panax ginseng CA Mey.) grown in Changbai Mountain for 3 years; the ginseng is weighed, with the ginseng root (including the rhizome) accounting for 70-85% of the weight, the ginseng leaves accounting for 10-15%; the rest are stems, petioles, and other parts.

[0069] Example 1: Detection of active ingredients in fresh ginseng.

[0070] According to the relevant methods under the Ginseng entry in Part I of the 2025 edition of the Chinese Pharmacopoeia, and the relevant methods under “T_CNHFA 001-2021_Determination of the Content of Rare Ginsenosides in Ginseng”, the contents of different saponins in Table 1 below were determined.

[0071] Table 1. Types of ginsenosides determined

[0072]

[0073] The content of ginsenosides in fresh ginseng leaves and ginseng roots is shown in Table 2.

[0074] Table 2. Content of 10 ginsenosides in ginseng root and ginseng leaf (mg / g)

[0075]

[0076] (2) Determination of total ginseng polysaccharides:

[0077] To prepare a standard curve for total polysaccharides: Accurately weigh 10 mg of glucose standard into a 10 mL volumetric flask, and dilute to the mark with distilled water to prepare a 1 mg / mL glucose standard solution. Add 0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of glucose standard solution to seven test tubes sequentially, then add distilled water to a final volume of 2 mL. Mix thoroughly. Then, add 0.5 mL of the standard solution at different concentrations to each test tube, followed by 0.5 mL of 6% phenol and 2.5 mL of 98% concentrated sulfuric acid. Allow the tubes to cool at room temperature for 30 min, and measure the absorbance at 490 nm. Use distilled water instead of the glucose solution as a blank control. Plot a standard curve with glucose concentration on the x-axis and absorbance on the y-axis.

[0078] Preparation of the test solution: Accurately weigh 0.5 g of ginseng powder, add 10 mL of distilled water, and extract twice by ultrasonication at 50 ℃ for 30 min. Combine the two filtrates to obtain the supernatant. Add anhydrous ethanol to the supernatant and precipitate overnight at 4 ℃. After centrifugation, add 50 mL of distilled water and dissolve the precipitate by ultrasonication at 50 ℃. Take 1 mL of the extract and dilute it 10 times. Determine the content of total ginseng polysaccharides using the phenol-sulfuric acid colorimetric method. Measure the absorbance at 490 nm using a UV spectrophotometer.

[0079] The total polysaccharide content of ginseng leaves and ginseng roots is shown in Table 3.

[0080] Table 3 Total polysaccharides from ginseng root and ginseng leaf (mg / g)

[0081]

[0082] (3) The volatile oil content of ginseng root was determined by steam distillation.

[0083] Take fresh ginseng and crush it through a No. 2 sieve;

[0084] Accurately weigh approximately 100g of ginseng powder (accurate to 0.01g) and record its exact mass (denoted as m).

[0085] Place the weighed sample powder into a 1000 mL round-bottom flask.

[0086] Add approximately 500 mL of distilled water and a magnetic stir bar to the flask.

[0087] Slowly add 5g of sodium chloride along the bottle wall to facilitate oil-water separation.

[0088] Connect the flask to the volatile oil analyzer, and then add an appropriate amount of distilled water to the analyzer so that the water level is slightly lower than the overflow port.

[0089] Finally, connect the condenser coil, ensuring all connections are properly sealed. Fill the condenser coil with water until it is almost overflowing.

[0090] Turn on the cooling water and keep the water flow stable.

[0091] Begin heating the round-bottom flask. Control the heating rate to maintain steady boiling, ensuring the distillate drips at a rate of approximately 2-3 drops per second.

[0092] Continue distilling for 5 hours, then stop heating and distilling until the volume of volatile oil in the measuring device no longer increases, and wait 15 minutes; allow the oil droplets adhering to the inner wall of the condenser to flow fully into the lower part of the measuring device.

[0093] After the oil and water phases in the measuring apparatus have completely separated into layers and become clear and transparent, the volume of volatile oil is read (denoted as V, unit: mL).

[0094] Volatile oil content (mL / 100g) = (V / m) × 100

[0095] Finally, the volatile oil content of ginseng was calculated to be 0.06 mL / 100g.

[0096] Example 2: Effects of different treatment methods on the active components of ginseng.

[0097] After washing the fresh ginseng, use a sterilized fine needle to create uniform micro-wounds on the surface of the ginseng (wound area approximately 10%, depth not exceeding 0.2mm); then immerse it completely in the stress solution for 30 minutes.

[0098] The processed fresh ginseng was placed in a high humidity (>90% RH), 20-25°C, and dark environment for 7 days for induction culture.

[0099] Then, the contents of ginsenosides, total ginseng polysaccharides, and volatile oils were tested according to the method in Example 1; the results are shown in Tables 4-1, 4-2, and 4-3.

[0100] The sample processing methods for different serial numbers are as follows:

[0101] 1. The stress fluid contains the following components:

[0102] The activated fluorescent Pseudomonas suspension has a concentration ≥10 6 CFU / mL;

[0103] Methyl o-hydroxybenzoate at a concentration of 500 μM;

[0104] The rest is deionized water.

[0105] 2: The stress fluid contains the following components:

[0106] The activated fluorescent Pseudomonas suspension has a concentration ≥10 6 CFU / mL;

[0107] The rest is deionized water.

[0108] 3: The stress fluid contains the following components:

[0109] Methyl o-hydroxybenzoate at a concentration of 500 μM;

[0110] The rest is deionized water.

[0111] 4: The stress fluid contains the following components:

[0112] The activated Bacillus subtilis suspension has a concentration ≥10 6 CFU / mL;

[0113] Methyl o-hydroxybenzoate at a concentration of 500 μM;

[0114] The rest is deionized water.

[0115] 5: The stress fluid contains the following components:

[0116] The activated Trichoderma viride suspension has a concentration ≥10 6 CFU / mL;

[0117] Methyl o-hydroxybenzoate at a concentration of 500 μM;

[0118] The rest is deionized water.

[0119] 6: The stress fluid contains the following components:

[0120] The activated Bacillus subtilis suspension has a concentration ≥10 6CFU / mL;

[0121] The rest is deionized water.

[0122] 7: The stress fluid contains the following components:

[0123] The activated Trichoderma viride suspension has a concentration ≥10 6 CFU / mL;

[0124] The rest is deionized water.

[0125] 8: The stress fluid contains the following components:

[0126] The activated fluorescent Pseudomonas suspension has a concentration ≥10 6 CFU / mL;

[0127] Methyljasmonic acid at a concentration of 500 μM;

[0128] The rest is deionized water.

[0129] 9: The stress fluid contains the following components:

[0130] Methyljasmonic acid at a concentration of 500 μM;

[0131] The rest is deionized water

[0132] 10: The stress fluid contains the following components:

[0133] The activated fluorescent Pseudomonas suspension has a concentration ≥10 6 CFU / mL;

[0134] Methyl o-hydroxybenzoate at a concentration of 100 μM;

[0135] The rest is deionized water.

[0136] 11: The stress solution is deionized water.

[0137] 12: Dry the fresh ginseng before soaking it in the stress solution.

[0138] Table 4-1 Content of active ingredients in different treatment groups (ginseng root, mg / g)

[0139]

[0140] Table 4-2 Content of active ingredients in different treatment groups (ginseng leaves, mg / g)

[0141]

[0142] Table 4-3 Content of active ingredients in different treatment groups (polysaccharides mg / g and volatile oil mL / 100g)

[0143]

[0144] As shown in the table above, *Pseudomonas fluorescens* can catalyze the conversion of saponins CK, Rh1, F1, Rg5, and Rk1, and the conversion effect is even higher when stressed simultaneously with the effective concentration of methyl o-hydroxybenzoate. *Trichoderma viride* also has some ability to catalyze the conversion of saponin F1, but the effect is far less than that of *Pseudomonas fluorescens*.

[0145] Example 3: Effect of culture time on the content of ginsenosides and ginseng polysaccharides.

[0146] According to the experimental results of Example 2, the effect of promoting active ingredients was best when fresh ginseng was simultaneously treated with *Pseudomonas fluorescens* and methyl phthalate. Using the treatment method in Example 2 (number 1), the total polysaccharide content and ginsenoside Rb1 content in ginseng roots were tested on days 1, 3, 7, 10, and 14 as representative samples; the specific effects of different culture times on the yield were studied; the results are shown in Table 5.

[0147] Table 5. Effects of different culture times on the content of active ingredients (ginseng root, mg / g)

[0148]

[0149] Example 4: Extraction of ginseng.

[0150] Extraction was performed on the ginseng processed in Example 2, number 1; the specific steps are as follows;

[0151] 1. Extraction from ginseng leaves:

[0152] S1: Cut the ginseng leaves from the induced culture fresh ginseng, place the ginseng leaves in a vacuum drying oven, and dry them to constant weight (moisture content ≤8%) at 50℃ and -0.08MPa.

[0153] The dried ginseng leaves are crushed and sieved (40 mesh) to obtain fine ginseng leaf powder for later use.

[0154] S2: Aqueous extracts of ginseng polypeptides and proteins:

[0155] Add ginseng leaf powder to pure water (solid-to-liquid ratio 1:20 g / mL); extract twice at 40℃; filter the water extract to obtain a supernatant containing polypeptides and ginseng leaf residue.

[0156] The supernatant containing peptides is concentrated under reduced pressure, and the polysaccharides in the aqueous extract are precipitated with alcohol. The polysaccharide fractions are combined and purified in subsequent steps. The supernatant after alcohol precipitation is separated, purified, and dried by gel chromatography to separate the peptide components.

[0157] S3: Ultrasonic-assisted ethanol extraction of ginseng leaf residue:

[0158] Extraction solvent: Prepare an aqueous ethanol solution with a concentration of 50 wt%.

[0159] Feeding: Accurately weigh 100g of ginseng leaf powder and put it into the ultrasonic reflux extraction device. Add ethanol solution at a material-to-liquid ratio of 1:15 (g / mL).

[0160] Extraction: Turn on the ultrasonic device (power set to 600W, frequency 25kHz), and simultaneously turn on the heating reflux device, controlling the temperature at 45±5℃ for extraction for 20 minutes.

[0161] Filtration and repeated extraction: After extraction, filter the liquid while it is still hot using a Buchner funnel and collect the filtrate. Return the filter residue to the extraction device and repeat the extraction once more under the same material-liquid ratio and conditions.

[0162] Combine the filtrates: Combine the filtrates from the two extractions to obtain the total extract.

[0163] S4 Concentration and Preliminary Purification:

[0164] Concentration under reduced pressure: The combined extracts were transferred to a rotary evaporator and concentrated under reduced pressure at a water bath of 60°C and a pressure of -0.08 MPa to recover ethanol until an alcohol-free fluid extract was obtained.

[0165] Centrifugation: Add a small amount of pure water to the fluid extract for dilution, then centrifuge at 8000 rpm for 15 minutes to remove insoluble impurities and collect the supernatant.

[0166] Separation and purification using S5 macroporous adsorption resin:

[0167] Resin pretreatment and column packing: AB-8 type macroporous adsorption resin (non-polar) was selected, soaked in ethanol and washed with water until there was no alcohol odor, and then wet-packed into a column (column diameter to height ratio 1:10) to prepare a resin column.

[0168] Sample loading: The supernatant after centrifugation is slowly loaded onto the resin column at a flow rate of 2 BV / h (column volume / hour).

[0169] Water elution (collection of polysaccharide components): After sample loading, elute with 5-7 BV of pure water at a flow rate of 4 BV / h. The eluent at this stage mainly contains ginseng polysaccharides.

[0170] The sulfuric acid-phenol method was used for follow-up detection, and positive fractions were collected.

[0171] Eluent elution (collection of saponin fractions): After water washing, elution was performed using 5 BV of 75% (v / v) ethanol solution at a flow rate of 2 BV / h. The eluent at this stage mainly contained ginsenosides. The fractions showing positive results were collected using the vanillin-perchloric acid method or TLC for monitoring and detection.

[0172] S6 Post-Processing and Refining:

[0173] Refining of ginseng polysaccharides:

[0174] Concentration: The water-eluted portion of the collected liquid is concentrated under reduced pressure at 60°C to 1 / 5 of its original volume.

[0175] Sevage method for deproteinization: Add 1 / 4 volume of chloroform-n-butanol mixture (V chloroform:V n-butanol = 4:1) to the concentrate, shake vigorously for 20 minutes, and centrifuge to remove the intermediate protein gel and the lower organic phase. Repeat this operation until no white interface is formed in the middle.

[0176] Alcohol precipitation: Slowly add 4 times the volume of 95% ethanol to the supernatant (to make the final ethanol concentration reach 80%), place it in a refrigerator at 4°C and let it stand overnight to allow the polysaccharide to precipitate completely.

[0177] Washing and drying: The precipitate was collected by centrifugation and washed twice each with anhydrous ethanol and acetone, respectively, to remove water and color. The precipitate was then placed in a vacuum freeze dryer and freeze-dried at -50°C for 24 hours to obtain a white flocculent ginseng polysaccharide product, which was then pulverized for later use.

[0178] Post-processing of ginsenosides:

[0179] Concentration: The alcohol-eluted portion was concentrated to dryness under reduced pressure at 60°C to obtain a brownish-yellow saponin extract.

[0180] Drying: The extract was transferred to a vacuum drying oven and dried at 60°C to constant weight to obtain a light yellow ginsenoside refined powder, which was then pulverized for later use.

[0181] 2. Extraction of ginseng root:

[0182] S1 preprocessing and pretreatment:

[0183] In step S1 of the extraction of ginseng leaves, the fresh ginseng after induction culture is cut to obtain ginseng roots, which are then washed, vacuum dried at low temperature, and pulverized through a 40-mesh sieve to obtain ginseng root powder.

[0184] S2 steam distillation method for extracting volatile oils:

[0185] Apparatus: The volatile oil extraction apparatus specified in the Chinese Pharmacopoeia is used.

[0186] Feeding: Weigh 500g of ginseng root powder, mix it evenly with an appropriate amount of pure water and soak it for 1 hour, then put it into a volatile oil extractor.

[0187] Distillation: Heat to a gentle boil and perform steam distillation. Continue to observe after 6 hours of distillation until the oil volume no longer increases.

[0188] Collection: After cooling, read the volume of volatile oil, carefully aspirate the upper layer of volatile oil with a pipette, add anhydrous sodium sulfate to dry and remove water, transfer to a brown sample bottle, seal with nitrogen, and store at 4°C in the dark for later use.

[0189] Residue treatment: Collect the remaining ginseng residue in the distillation vessel, drain the water, and dry it at 60°C for the next extraction step.

[0190] S3: Aqueous extracts of ginseng polypeptides and proteins:

[0191] Add ginseng residue to pure water (solid-to-liquid ratio 1:20 g / mL); add papain at 0.2% of the ginseng residue mass and hydrolyze at 50℃ for 1 hour; then extract twice at 40℃.

[0192] The aqueous extract was filtered to obtain a supernatant containing polypeptides and a secondary residue.

[0193] The supernatant containing peptides is concentrated under reduced pressure, and the polysaccharides in the aqueous extract are precipitated with alcohol. The polysaccharide fractions are combined and purified in subsequent steps. The supernatant after alcohol precipitation is separated, purified, and dried by gel chromatography to separate the peptide components.

[0194] Ultrasonic-assisted extraction and subsequent purification of S4 secondary residue

[0195] Extraction: Same as steps S2 and S3 for ginseng leaf extraction. The dried secondary residue was extracted twice under the same material-to-liquid ratio (1:18 g / mL), solvent concentration (65% ethanol), temperature (45℃), and ultrasonic conditions. The filtrates were combined and concentrated under reduced pressure to obtain a fluid extract. The supernatant was collected by centrifugation.

[0196] Resin purification: Same as step S4 of ginseng leaf extraction. Load the supernatant onto an AB-8 resin column, first elute with water to collect the ginseng polysaccharide fraction, then elute with 75% ethanol to collect the ginsenoside fraction.

[0197] Post-processing: This step is the same as step S5 in the extraction of ginseng leaves. The water-eluted fraction is subjected to deproteinization, alcohol precipitation, and freeze-drying to obtain ginseng root polysaccharides; the alcohol-eluted fraction is concentrated and vacuum-dried to obtain ginseng root saponins.

[0198] Based on 100g of ginseng root and ginseng leaves, the extraction yield of this invention is:

[0199] Ginseng leaf-ginseng polysaccharide fraction: 76.88 mg / g;

[0200] Ginseng leaf extract - ginsenoside fraction: 46.5 mg / g;

[0201] Ginseng leaf - polypeptide content: 45.5 mg / g;

[0202] Ginseng root-ginseng polysaccharide fraction: 29.8 mg / g;

[0203] Ginseng root-ginsenoside fraction: 25.81 mg / g;

[0204] Ginseng root polypeptide content: 20.1 mg / g;

[0205] Ginseng root - volatile oil: 0.06mL / 100g.

[0206] The peptide content was measured using the Kjeldahl method.

[0207] Since the extract in this embodiment also contains ginsenosides or polysaccharides that were not detected in this invention, its extraction amount will be higher than the detection amount in the aforementioned embodiments.

[0208] Example 5: Preparation of ginseng stress extract.

[0209] The ginseng leaf polypeptides, ginseng leaf polysaccharides, ginseng leaf saponins, ginseng root polypeptides, ginseng root volatile oil, ginseng root polysaccharides, and ginseng root saponins obtained in Example 4 were added to phospholipids and deionized water; a stable nanoemulsion was prepared by high-pressure homogenization; the total concentration of the active ingredients was 15%; and ginseng stress extract was obtained.

[0210] The mass ratio of ginseng leaf (ginseng leaf polysaccharides, ginseng leaf saponins, ginseng leaf polypeptides) to ginseng root (ginseng root polypeptides, ginseng root volatile oil, ginseng root polysaccharides, ginseng root saponins) is 1:7. Since ginseng leaf polysaccharides and ginseng leaf saponins originate from the same batch of ginseng leaf raw materials, and ginseng root volatile oil, ginseng root polysaccharides, and ginseng root saponins originate from the same batch of ginseng root raw materials, the proportions of these raw materials will not be adjusted; they will be blended according to the yield of each raw material from the same batch.

[0211] Example 6 Performance testing of ginseng stress extract

[0212] 1. Anti-inflammatory experiments were conducted using LPS-induced RAW264.7 macrophages.

[0213] Test method:

[0214] The system included a blank control group, a model group, a positive control group, and an experimental group. Each well in the blank control group was added with complete culture medium. Each well in the positive control group was added with 5 μM dexamethasone sodium acetate diluted with complete culture medium. Each well in the experimental group was added with the sample diluted with complete culture medium.

[0215] Each group was set up with 3 replicates and incubated at 37℃ in a 5% CO2 incubator for 2 h. Except for the blank control group, LPS was added to each well to a final concentration of 1 μg / mL and incubated at 37℃ in a 5% CO2 incubator for 24 h. The cell supernatant was collected by centrifugation at low temperature and detected according to the NO detection kit instructions. The NO content in the collected cell supernatant was measured, and the experimental data are shown in Table 6.

[0216] Experimental group: Samples diluted with complete culture medium and LPS; the samples were from Example 5 and diluted 10 times, with a final active ingredient concentration of approximately 1.5%.

[0217] Model group: complete culture medium and LPS;

[0218] Positive control group: 5 μM dexamethasone acetate and LPS diluted with complete culture medium;

[0219] Blank control group: complete culture medium only.

[0220] Table 6 Results of anti-inflammatory experiments

[0221]

[0222] Collagenase activity inhibition experiment

[0223] Experimental methods:

[0224] Prepare 1 mg / mL tetracycline hydrochloride (positive control) and test sample solutions using Tris-HCl buffer (pH = 7.5 ± 0.2, containing 5 mmol / mL CaCl2); prepare 0.5 mg / mL substrate FALGPA buffer solution; and prepare 0.2 mg / mL type I collagenase Tris-HCl solution.

[0225] A blank control group, an experimental group, and a sample blank group were set up. 140 μL of 0.2 mg / mL enzyme solution was added to the control and experimental groups, while an equal volume of buffer solution was added to the blank and sample blank groups. After incubating at 37°C for 20 min, 40 μL of FALGPA substrate solution was added to each group. After incubating at 37°C for 20 min, the absorbance change of each component at 330 nm was measured. Each group was measured three times on average, and the average value was taken. The inhibition rate of collagenase was calculated according to the following formula, and the results are shown in Table 7.

[0226] Sample blank group: 60 μL sample solution + 140 μL buffer salt + 40 μL substrate

[0227] Experimental group: 60 μL sample solution + 140 μL collagenase + 40 μL substrate

[0228] Control group: 60 μL buffer salt + 140 μL collagenase + 40 μL substrate

[0229] Control group: 200 μL buffer + 40 μL substrate

[0230] The sample solution was from Example 5 and diluted 10 times, resulting in a final active ingredient concentration of approximately 1.5%.

[0231]

[0232] Table 7 Results of collagenase activity inhibition experiment

[0233]

[0234] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. A ginseng stress extract, characterized in that, The preparation of the ginseng stress extract includes the following steps: S1 Pretreatment: Fresh ginseng is washed and then soaked in stress solution for treatment; The stress fluid contains the following components: The activated fluorescent Pseudomonas suspension has a concentration ≥10 6 CFU / mL; S2 induction culture: induction culture of pretreated fresh ginseng; Induction conditions are: >90% RH, 20-25℃; protected from light; S3 Sterilization and Separation: The ginseng after induction culture is sterilized and then separated into two parts: ginseng leaves and ginseng roots; S4 Ginseng Leaf Extract: Ginseng leaves are dried, and then ginseng polypeptides, ginseng polysaccharides and ginsenosides are extracted and purified separately for later use; S5 Ginseng Root Extract: Ginseng roots are dried and extracted and purified in the following order: volatile oil, ginseng polypeptides, ginsenosides, and ginseng polysaccharides; for later use. S6 Mixing: Mix the components extracted in steps S4 and S5; this is the ginseng stress extract. In step S1, the stress solution also contains methyl o-hydroxybenzoate at a concentration of ≥500 μM. The induction culture time in step S2 is at least 3 days; The step S4 ginseng leaf extraction is as follows: the ginseng leaves are dried and passed through a sieve of at least 40 mesh, first extracted with pure water, and the supernatant and residue are separated; the residue is extracted with an ethanol aqueous solution; after extraction, the extract is concentrated, and the concentrate is eluted with resin to remove ginseng polysaccharides and ginsenosides; The step S5 ginseng root extraction is as follows: the ginseng root is dried and passed through a sieve of at least 40 mesh, and the ginseng root powder is processed by steam distillation to obtain volatile oil and ginseng residue; Water was added to the ginseng residue and enzymatic hydrolysis was performed using protease; after enzymatic hydrolysis, solid-liquid separation was performed to obtain a polypeptide aqueous extract and secondary residue; The secondary residue was extracted with an ethanol-water solution; after extraction, the extract was concentrated, and the concentrate was eluted with resin to remove ginseng polysaccharides and ginsenosides.

2. The ginseng stress extract according to claim 1, characterized in that, In step S4, ginseng leaf extraction, the residue is extracted with ultrasound assistance, the ethanol concentration in the aqueous ethanol solution is at least 50 wt%, the material-to-liquid ratio during extraction is 1:20 g / mL, reflux extraction is performed at 40-50℃, and the extraction is performed at least twice, each time for at least 20 minutes. When the concentrate is made of resin, ginseng polysaccharides are first eluted with pure water; then ginsenosides are eluted with an ethanol solution of ≥70% concentration. The supernatant was concentrated under reduced pressure and precipitated with alcohol to obtain ginseng polysaccharide and ginseng polypeptide solutions. The ginseng polypeptide solutions were purified by chromatographic separation and then dried. After elution, ginseng polysaccharides were concentrated, deproteinized, precipitated with alcohol, washed, and dried. Ginsenosides were concentrated and dried after elution.

3. The ginseng stress extract according to claim 1, characterized in that, In step S5, ginseng root extraction, the secondary residue is extracted with ultrasonic-assisted extraction using an ethanol-water solution, wherein the ethanol concentration in the ethanol-water solution is at least 50 wt%; the material-to-liquid ratio during extraction is 1:18 g / mL; reflux extraction is performed at 40-50℃; at least twice, each time for at least 20 minutes. When the concentrate is made of resin, ginseng polysaccharides are first eluted with pure water; then ginsenosides are eluted with an ethanol solution of ≥70% concentration. The aqueous extract of the polypeptide was concentrated under reduced pressure, filtered through an ultrafiltration membrane to obtain the filtrate, and the filtrate was precipitated with alcohol to obtain ginseng polysaccharide and ginseng polypeptide solution. The ginseng polypeptide solution was purified by chromatographic separation and then dried. After elution, ginseng polysaccharides were concentrated, deproteinized, precipitated with alcohol, washed, and dried. Ginsenosides were eluted, concentrated, and dried.

4. The use of the ginseng stress extract according to any one of claims 1-3 in the preparation of cosmetics.

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