An enzyme composition for producing rare ginsenosides and its application

Abstract

The present application relates to the field of medical technology, and specifically discloses an enzyme composition for generating rare ginsenosides and its application. The enzyme composition includes pectin methylesterase and β-glycosidase; the mass ratio of the pectin methylesterase and β-glycosidase is 1: (0.2~2). The present application adopts pectin methylesterase and β-glycosidase to cooperate with each other, which can efficiently convert ginseng stem and leaf extract or ginsenoside Rb1 into rare ginsenoside CK, with high conversion efficiency, low cost, simple operation and environmental protection. And the rare ginsenosides obtained by the enzymatic hydrolysis method have the advantages of high selectivity, mild reaction conditions, high conversion rate, easy separation and purification, no pollution to the environment and low cost. The rare ginsenosides generated by the enzyme composition of the present application show stronger skin repair effects, can be better used in skin care products, and have good application prospects.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and in particular to an enzyme composition for producing rare ginsenosides and its application. Background Art

[0002] Ginsenoside, also known as ginsenoside, is a tetracyclic triterpenoid saponin extracted from Araliaceae plants. Ginsenoside has the activities of relieving fatigue, improving memory, anti-aging, removing free radicals in the body, regulating the central nervous system of the brain, and anti-cancer, and has become a hot field in natural medicine research. Studies have found that the secondary metabolite derivatives of ginsenosides after transformation have stronger biological activity. This secondary metabolite derivative is named "rare ginsenoside", and the ginsenoside extracted directly from Araliaceae plants is called "prototype ginsenoside". At present, more than 60 rare ginsenosides have been discovered, including CK, F2, Rk2, Rg3, Rh2, Rg5, Rh1, Rh3, Rk1 and other rare ginsenosides with different biological activities. The content of these rare ginsenosides in plants is extremely low, and simple separation and extraction is difficult to meet application needs.

[0003] Rare ginsenosides can effectively neutralize free radicals, slow down oxidative damage, and protect the skin from environmental damage; stimulate the synthesis of collagen, improve skin elasticity and firmness, and reduce the formation of wrinkles and fine lines; effectively relieve skin inflammation and redness; inhibit melanin production, reduce spots and freckles, and brighten skin tone; dilate blood vessels, increase blood flow, and promote skin metabolism and repair ability.

[0004] At present, rare ginsenosides can be obtained through methods such as chemical reactions and biotransformations. However, the chemical method for converting rare saponins has the disadvantages of difficult-to-control reaction conditions, complex products, and environmental pollution. Therefore, the process and method for preparing rare ginsenosides by biotransformation has attracted continuous attention from researchers. However, using the current biotransformation method, the bioconversion rate of prototype ginsenosides into rare ginsenosides is low and the conversion cost is high. Summary of the invention

[0005] The purpose of this application is to overcome the shortcomings of the above-mentioned prior art and provide an enzyme composition for generating rare ginsenosides and its application. The enzyme composition designed by this application can efficiently transform and synthesize rare ginsenosides. The rare ginsenosides obtained by transformation show stronger skin repair effects and have broad market prospects for application in cosmetics.

[0006] To achieve the above purpose, the technical solution adopted by this application is:

[0007] The present application provides an enzyme composition for producing rare ginsenosides, the enzyme composition comprising pectin methylesterase and β-glucosidase;

[0008] The mass ratio of the pectin methylesterase to the β-glycosidase is 1:(0.2-2).

[0009] In the technical solution of the present application, the present application uses pectin methylesterase and β-glucosidase to cooperate with each other, which can efficiently convert the conversion precursor into rare ginsenosides, with high conversion efficiency, low cost, simple operation and environmental protection and no pollution. The rare ginsenosides converted by the enzyme composition of the present application show stronger skin repair effect, can be better used in skin care products, and have good application prospects.

[0010] Among them, the present application uses a combination of pectin methylesterase and β-glycosidase in a mass ratio of 1: (0.2~2), which can better promote the conversion of precursor substances into rare ginsenosides, with a higher conversion efficiency and a higher content of rare ginsenosides obtained.

[0011] As a preferred embodiment of the enzyme composition for producing rare ginsenosides described in the present application, the mass ratio of pectin methylesterase to β-glycosidase is 1:(0.5~1).

[0012] When the pectin methylesterase and β-glycosidase of the present application are used in the above mass ratio, the conversion effect of producing rare ginsenosides is better, the conversion rate is high, and the content of rare ginsenosides obtained is higher.

[0013] The present application also provides the use of the enzyme composition in producing rare ginsenosides.

[0014] The present application adopts enzyme combination and other enzymatic hydrolysis methods to obtain rare ginsenosides, which has the advantages of high selectivity, mild reaction conditions, high conversion rate, easy separation and purification, no pollution to the environment and low cost.

[0015] As a preferred embodiment of the application described in the present application, the rare ginsenoside includes rare ginsenoside CK.

[0016] As a preferred embodiment of the application described in the present application, the application is the application of using the enzyme composition to promote the conversion of precursor substances into rare ginsenosides.

[0017] The present application uses an enzyme composition composed of pectin methylesterase and β-glucosidase to convert the precursor into rare ginsenoside CK, which is simple to operate, has obvious effects, and has low conversion costs. The rare ginsenosides after conversion show stronger skin repair effects and have good application prospects.

[0018] As a preferred embodiment of the application described in the present application, the mass ratio of the enzyme composition to the conversion precursor is (0.5-10):1, preferably (1-4):1.

[0019] The present application adopts the enzyme composition and conversion precursor substance within the above specific mass ratio range, so that the enzyme composition can better promote the conversion precursor substance to produce rare ginsenosides, and the production amount of rare ginsenosides is higher.

[0020] As a preferred embodiment of the application described in the present application, the conversion precursor substance includes ginseng stem and leaf extract or ginsenoside Rb1.

[0021] The enzyme composition of the present application can efficiently convert ginseng stem and leaf extract or ginsenoside Rb1 into rare ginsenoside CK, with high conversion efficiency, low cost, simple operation and environmental protection. After conversion, rare ginsenoside CK shows stronger skin repair effect and has broad market prospects for application in cosmetics.

[0022] As a preferred embodiment of the application described in this application, the conversion step includes:

[0023] The enzyme composition is used to promote the conversion of precursor substances into rare ginsenosides under the conditions of a conversion temperature of 40-60° C., a conversion time of 18-48 hours, and a pH value of 4.0-6.0.

[0024] Preferably, the conversion temperature is 45-55° C., the conversion time is 24-48 hours, and the pH value is 4.5-5.5.

[0025] Under the above-mentioned conversion conditions, the enzyme composition of the present application can better and efficiently convert ginseng stem and leaf extract or ginsenoside Rb1 into rare ginsenoside CK, with high conversion efficiency and high production of rare ginsenoside CK.

[0026] Preferably, after the conversion is completed, the converted solution is kept at 90° C. for 30 minutes and then filtered to obtain rare ginsenosides.

[0027] The present application also provides a method for producing rare ginsenosides, which uses the enzyme composition to promote the conversion of ginseng stem and leaf extract or ginsenoside Rb1 into rare ginsenosides.

[0028] The present application promotes the conversion of precursor substances to obtain rare ginsenosides by an enzymatic hydrolysis method (adding the enzyme composition of the present application). The method of generating rare ginsenosides by using the enzyme composition of the present application has the advantages of high selectivity, mild reaction conditions, high conversion rate, easy separation and purification, no pollution to the environment and low cost.

[0029] Preferably, the method for producing rare ginsenosides comprises the following steps:

[0030] The ginseng stem and leaf extract or ginsenoside Rb1 was weighed and dissolved in acetic acid / sodium acetate buffer. After adding the enzyme composition, the hydrolysis conversion was carried out under the conditions of a conversion temperature of 40-60°C and a pH value of 4.0-6.0. The conversion time was 18-48 hours. After the conversion was completed, the reaction was stopped by incubation at 90°C for 30 minutes. N-butanol was added for extraction. The n-butanol phase was evaporated to dryness under reduced pressure and redissolved in anhydrous ethanol. The conversion result was detected by HPLC.

[0031] The present application also provides the use of the enzyme composition to generate rare ginsenosides in skin care products.

[0032] This application uses pectin methylesterase and β-glucosidase to efficiently convert ginseng stem and leaf extract or ginsenoside Rb1 into rare ginsenoside CK, with high conversion efficiency, low cost, simple operation and environmental protection. After conversion, rare ginsenoside CK shows stronger skin repair effect and has broad market prospects for application in cosmetics.

[0033] Compared with the prior art, this application has the following beneficial effects:

[0034] The present application provides an enzyme composition for generating rare ginsenosides and its application. The present application uses pectin methylesterase and β-glycosidase to cooperate with each other, which can efficiently convert ginseng stem and leaf extract or ginsenoside Rb1 into rare ginsenoside CK, with high conversion efficiency, low cost, simple operation and environmental protection. The rare ginsenosides obtained by the enzymatic hydrolysis method have the advantages of high selectivity, mild reaction conditions, high conversion rate, easy separation and purification, no pollution to the environment and low cost. The rare ginsenosides generated by the enzyme composition of the present application show stronger skin repairing effects, can be better used in skin care products, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the result diagram of the effect of enzymatic hydrolysis on the content of rare ginsenoside CK in the system;

[0036] Figure 2 This is a graph showing the effect of rare ginsenoside CK obtained in Example 30 and Comparative Examples 1-2 on the system's inhibition of matrix metalloproteinase-1 activation. DETAILED DESCRIPTION

[0037] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0038] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are of the same type.

[0039] In the following examples and comparative examples, pectin methylesterase was purchased from Ningxia Xiasheng Industrial Group Co., Ltd.;

[0040] β-Glucosidase was purchased from Amano Enzyme Products, Aromase H2;

[0041] Ginsenoside Rb1, purity 55%, commercially available;

[0042] Ginsenoside CK standard, purity ≥98%, commercially available;

[0043] FALGPA, purity >97%, commercially available.

[0044] Preparation of ginseng stem and leaf extract: Grind ginseng leaves and stems, pass through a 60-mesh sieve, weigh 200 g, add 4 L methanol, perform ultrasonic extraction (power 100 Hz / temperature 45 ° C), filter with a 0.45 μm filter membrane to obtain the supernatant, and repeat the extraction of the residue three times according to the above process. Combine the supernatants, concentrate under reduced pressure, and dry to obtain a crude ginseng stem and leaf extract. The crude extract is defatted three times with petroleum ether, dried, dissolved in water, and then depigmented with activated carbon three times. After filtering, extract it three times with 600 mL of n-butanol, combine the n-butanol layers, evaporate to dryness, and obtain the ginseng stem and leaf extract, which is used as a substrate for enzymatic hydrolysis.

[0045] Examples 1-4 and Comparative Examples 1-4: Effects of different enzyme compositions on the conversion of rare ginsenoside CK

[0046] Conversion of rare ginsenoside CK: Weigh 50 mg of ginseng stem and leaf extract as substrate, dissolve in 10 mL of acetic acid / sodium acetate buffer, add 50 mg of the enzyme composition in Table 1 (the mass ratio of the enzyme composition to the substrate is 1:1) or enzyme, and hydrolyze at 45°C. The pH value of the reaction is 4.5. The conversion takes 24 hours. After the end, incubate at 90°C for 30 minutes to stop the reaction. Add an equal amount of n-butanol for extraction, evaporate the n-butanol phase under reduced pressure, and redissolve in anhydrous ethanol. The conversion result is detected by HPLC. The result of Example 4 is shown in the figure below. Figure 1 As shown (the system without enzyme addition means that no enzyme is added).

[0047] HPLC detection method: C18 chromatographic column (250 mm × 4.6 mm, 5 μm), mobile phase (acetonitrile A: 0.05% phosphoric acid water B), gradient elution (0-10 minutes 50% A, 10-25 minutes 70% A), column temperature 25 ° C, detection wavelength 203 nm, flow rate 1 mL / min, injection volume 20 μl.

[0048] Table 1

[0049]

[0050] Examples 5-9 and Comparative Examples 6-7: Conversion of rare ginsenoside CK using enzyme compositions and substrates in different ratios Impact

[0051] Conversion of rare ginsenoside CK: Weigh 50 mg of ginseng stem and leaf extract as a substrate, dissolve it in 10 mL of acetic acid / sodium acetate buffer, add the enzyme composition obtained in Example 4 according to the addition amount in Table 2, and hydrolyze at 45°C. The pH value of the reaction is 4.5. The conversion takes 24 hours. After the reaction is completed, incubate at 90°C for 30 min to stop the reaction, add an equal amount of n-butanol for extraction, evaporate the n-butanol phase under reduced pressure, redissolve it in anhydrous ethanol, and detect the conversion result by HPLC.

[0052] The HPLC detection method is shown in Examples 1 to 4.

[0053] Table 2

[0054]

[0055] Examples 10-16: Effects of different conversion temperatures on the conversion of rare ginsenoside CK

[0056] Conversion of rare ginsenoside CK: 50 mg of ginseng stem and leaf extract was weighed as a substrate, dissolved in 10 mL of acetic acid / sodium acetate buffer, and 200 mg of the enzyme composition obtained in Example 4 (the mass ratio of the enzyme composition to the substrate was 4:1) was added, and then hydrolyzed at different conversion temperatures (as shown in Table 3). The pH value of the reaction was 4.5, and the conversion took 24 hours. After the end, the reaction was incubated at 90°C for 30 min to stop the reaction, and an equal amount of n-butanol was added for extraction. The n-butanol phase was evaporated to dryness under reduced pressure and redissolved in anhydrous ethanol. The conversion result was detected by HPLC.

[0057] The HPLC detection method is shown in Examples 1 to 4.

[0058] Table 3

[0059]

[0060] Examples 17-23: Effect of different pH values ​​on the conversion of rare ginsenoside CK

[0061] Conversion of rare ginsenoside CK: 50 mg of ginseng stem and leaf extract was weighed as a substrate, dissolved in 10 mL of acetic acid / sodium acetate buffer, and 200 mg of the enzyme composition obtained in Example 4 (the mass ratio of the enzyme composition to the substrate was 4:1) was added and hydrolyzed at 50°C. Different pH values ​​of the reaction (as shown in Table 4) were used for the conversion for 24 hours. After the reaction was stopped, the reaction was incubated at 90°C for 30 min, and an equal amount of n-butanol was added for extraction. The n-butanol phase was evaporated to dryness under reduced pressure and redissolved in anhydrous ethanol. The conversion result was detected by HPLC.

[0062] The HPLC detection method is shown in Examples 1 to 4.

[0063] Table 4

[0064]

[0065] Examples 24-29: Effects of different transformation times on the transformation of rare ginsenoside CK

[0066] Conversion of rare ginsenoside CK: 50 mg of ginseng stem and leaf extract was weighed as a substrate, dissolved in 10 mL of acetic acid / sodium acetate buffer, and 200 mg of the enzyme composition obtained in Example 4 (the mass ratio of the enzyme composition to the substrate was 4:1) was added and hydrolyzed at 50°C. The pH value of the reaction was 5.0, and the conversion was different (as shown in Table 5). After the reaction was stopped, the reaction was incubated at 90°C for 30 min, and an equal amount of n-butanol was added for extraction. The n-butanol phase was evaporated to dryness under reduced pressure, redissolved in anhydrous ethanol, and the conversion result was detected by HPLC.

[0067] Table 5

[0068]

[0069] Example 30: Effects of different substrates on the conversion of rare ginsenoside CK

[0070] Conversion of rare ginsenoside CK: Weigh 50 mg of ginsenoside Rb1 (55%) as a substrate, dissolve it in 10 mL of acetic acid / sodium acetate buffer, add 200 mg of the enzyme composition obtained in Example 4 (the mass ratio of the enzyme composition to the substrate is 4:1), and then hydrolyze at 50°C. The pH value of the reaction is 5.0, and the conversion time is 36 hours. After the reaction is completed, incubate at 90°C for 30 min to stop the reaction, add an equal amount of n-butanol for extraction, evaporate the n-butanol phase under reduced pressure, and redissolve it in anhydrous ethanol. The conversion results are detected by HPLC. The results are shown in Table 6.

[0071] Table 6

[0072]

[0073] It can be seen from the data in Tables 1-6 that the present application uses a synergistic combination of pectin methylesterase and β-glycosidase to convert ginseng stem and leaf extract or ginsenoside Rb1 into rare ginsenoside CK. The operation is simple, the effect is obvious, and the conversion cost is low. In particular, when pectin methylesterase and β-glycosidase are combined in a mass ratio of 1: (0.2~2), the conversion effect is better.

[0074] Example 31: Skin repair efficacy verification experiment

[0075] Human skin fibroblasts NHDF were seeded into 96-well culture plates at a cell density of 5000 cells / well. The culture medium was DMEM cell culture medium containing 10% fetal bovine serum. Each group had 3 replicate wells and was cultured in an incubator at 37°C, 5% CO2, and 95% humidity for 48 hours.

[0076] The cells were divided into: blank group (no UVA irradiation group); 200 kJ / m 2 UVA irradiation control group; 200 kJ / m 2 UVA irradiation test group. Comparative Examples 1 and 2 were prepared with acetic acid / sodium acetate buffer solution (2%), and Example 25 was prepared with acetic acid / sodium acetate buffer solution with different concentrations of dilutions (2%, 1% and 0.2%). Two hours before irradiation, 4% acetic acid / sodium acetate buffer solution was added to the blank group and the irradiation control group, and 4% dilutions of Comparative Example 1, Comparative Example 2 and Example 30 were added to the irradiation experimental group. After incubation for 2 hours, the supernatant was discarded from all groups, washed with PBS, and then a small amount of PBS was added to cover the cells for irradiation. The blank group was covered with tin foil. After irradiation, the culture medium containing 4% acetic acid / sodium acetate buffer solution or 4% dilutions of Comparative Example 1, Comparative Example 2 and Example 30 was continued to be added to the cells. After continuing to culture for 24 hours, the supernatant was collected and the activity of matrix metalloproteinase-1 (MMP-1) was detected. MMP-1 is an enzyme that hydrolyzes collagen. Excessive activation can cause skin elasticity loss and wrinkle formation.

[0077] Table 7

[0078]

[0079] Refer to the reagent addition amount in Table 7, add 50μL sample solution to the sample reaction well (A1) and sample background well (A0). Add 50μL cell culture medium containing 4% acetic acid / sodium acetate buffer to the solvent reaction well (B1) and solvent background well (B0). Then add 50μTricine buffer (50 mmol / L, pH 7.5) to the sample reaction well and sample background well, as well as the solvent reaction well and solvent background group. Add 50μL substrate FALGPA solution (1mmol / L, prepared in Tricine buffer) to the sample reaction well and solvent reaction well, and add 50μL Tricine buffer to the sample background group and solvent background group, shake the plate for 1min, incubate at 25℃ for 15min, and measure the absorbance at 335nm.

[0080] MMP-1 relative activity = (A1-A0) / (B1-B0)*100%

[0081] Table 8

[0082]

[0083] Through Table 8 and Figure 2 The results showed that the rare ginsenosides after transformation showed stronger inhibition of UVA-induced MMP-1 activation in human skin fibroblasts and had stronger skin repair effects.

[0084] The present application adopts enzyme combination and other enzymatic hydrolysis methods to obtain rare ginsenosides, which has the advantages of high selectivity, mild reaction conditions, high conversion rate, easy separation and purification, no pollution to the environment and low cost.

[0085] The present application also adopts the above-mentioned conversion conditions, which can better and efficiently convert ginseng stem and leaf extract or ginsenoside Rb1 into rare ginsenoside CK, with high conversion efficiency and high production of rare ginsenoside CK.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. Use of an enzyme composition in the production of rare ginsenoside CK, characterized in that: The enzyme composition is pectin methylesterase and β-glucosidase; Pectin methylesterase was derived from Aspergillus niger, and β-glycosidase was derived from Penicillium multicolor; The mass ratio of pectin methylesterase to β-glycosidase is 1:(0.2~2); The application is to use the enzyme composition to promote the conversion of precursor substances into rare ginsenoside CK; The transformation precursor is ginseng stem and leaf extract or ginsenoside Rb1; The preparation method of the ginseng stem and leaf extract comprises the following steps: The ginseng leaves and stems were crushed, passed through a 60-mesh sieve, 200 g was weighed, added to 4 L of methanol, and ultrasonic extraction was performed at a power of 100 Hz and a temperature of 45°C. The supernatant was filtered through a 0.45 μm filter membrane to obtain the supernatant. The residue was extracted 3 times according to the above process, and the supernatants were respectively combined, concentrated under reduced pressure, and dried to obtain a crude extract of ginseng stems and leaves; the crude extract of ginseng stems and leaves was defatted 3 times with petroleum ether, dried, dissolved in water, and then depigmented 3 times with activated carbon. After filtering, it was extracted 3 times with 600 mL of n-butanol, the n-butanol layers were combined, and evaporated to obtain a ginseng stem and leaf extract; The steps of the conversion include: The enzyme composition is used to promote the conversion of the precursor substance into rare ginsenoside CK under the conditions of a conversion temperature of 40-60° C., a conversion time of 18-48 hours, and a pH value of 4.0-6.

0.

2. The use according to claim 1, characterized in that The mass ratio of the pectin methylesterase to the β-glycosidase is 1:(0.5-1).

3. The use according to claim 1, characterized in that The mass ratio of the enzyme composition to the conversion precursor is (0.5-10):

1.

4. A method for producing rare ginsenoside CK, characterized in that: Using an enzyme composition to promote the conversion of ginseng stem and leaf extract or ginsenoside Rb1 into rare ginsenoside CK; The enzyme composition is pectin methylesterase and β-glucosidase; Pectin methylesterase was derived from Aspergillus niger, and β-glycosidase was derived from Penicillium multicolor; The mass ratio of pectin methylesterase to β-glycosidase is 1:(0.2~2); The steps of the conversion include: The enzyme composition is used to promote the conversion of ginseng stem and leaf extract or ginsenoside Rb1 into rare ginsenoside CK under the conditions of a conversion temperature of 40 to 60° C., a conversion time of 18 to 48 hours, and a pH value of 4.0 to 6.0; The preparation method of the ginseng stem and leaf extract comprises the following steps: The ginseng leaves and stems were crushed, passed through a 60-mesh sieve, 200 g was weighed, added to 4 L of methanol, and ultrasonic extraction was performed at a power of 100 Hz and a temperature of 45°C. The supernatant was filtered through a 0.45 μm filter membrane to obtain the supernatant. The residue was extracted three times according to the above process, and the supernatants were combined respectively, concentrated under reduced pressure, and dried to obtain a crude ginseng stem and leaf extract; the crude ginseng stem and leaf extract was defatted three times with petroleum ether, dried, dissolved in water, depigmented three times with activated carbon, filtered, extracted three times with 600 mL of n-butanol, the n-butanol layers were combined, and evaporated to obtain a ginseng stem and leaf extract.

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