Separation preparation method and application of non-natural ginsenoside

Abstract

The invention belongs to the technical field of medicine separation and preparation, and relates to a separation and preparation method and application of non-natural ginsenoside. The method comprises the following steps: extracting and concentrating a saccharomyces cerevisiae engineering bacterium fermentation product sample serving as a raw material to obtain a saponin crude extract I; enriching the saponin crude extract I by adopting macroporous adsorption resin to obtain a saponin crude extract II; and separating by adopting a high-speed counter-current chromatography to obtain a saponin extract III, namely the non-natural ginsenoside. The non-natural ginsenoside is a non-natural ginsenoside 3 [beta]-O-Glc-DM or a non-natural ginsenoside 20 [S]-O-Glc-DM, and the non-natural ginsenoside is a non-natural ginsenoside 3 [beta]-O- The method is simple, convenient and efficient, the prepared non-natural ginsenoside has the effects of reducing the activity of tyrosinase in cells and inhibiting melanogenesis, and a thought is provided for development and application of ginsenoside.

Description

Technical Field

[0001] The invention belongs to the technical field of drug separation and preparation, and in particular relates to a separation and preparation method and application of non-natural ginsenosides. Background Art

[0002] Ginsenosides are the main active ingredients of ginseng, mainly including dammarane saponins and oleanolic acid saponins. Dammarane ginsenosides use dammarenediol-II (DM) as a precursor and can biosynthesize two aglycones, protopanaxadiol (PPD) and protopanaxatriol (PPT). The C3 and C20 hydroxyl groups of PPD and the C6 and C20 hydroxyl groups of PPT are modified by glycosylation to produce a wide variety of ginsenosides, but DM glycosides are rarely identified from ginseng. Prototype ginsenosides are metabolized by enzymes and converted into rare ginsenosides, which have high biological activity and are more easily absorbed by the human body. In the prior art, UGTPg1 can catalyze the glycosylation of the C20 hydroxyl group of protopanaxadiol, and the yield of the rare non-natural ginsenoside 20S-O-Glc-DM in the fermentation product of the constructed UGTPg1 brewer's yeast engineered bacteria is significantly increased to 5.6 g / L; PgUGT74AE2 can catalyze the glycosylation of the C3 hydroxyl group of protopanaxadiol, and the PgUGT74AE2 brewer's yeast engineered bacteria is constructed. The engineered bacteria are fermented at a high density by batch feeding, and the yield of the rare non-natural ginsenoside 3β-O-Glc-DM in the fermentation product can reach 2.4 g / L. Due to the large amount of by-products produced during the fermentation process, the difficulty of separating the compounds is increased, so the effective separation of non-natural ginsenosides has not been achieved in the prior art. Since the development and utilization of non-natural ginsenosides generally require high-purity monomer compounds, a fast and efficient method is needed to effectively separate the active ingredients.

[0003] High-speed countercurrent chromatography belongs to liquid-liquid partition chromatography, which uses the polarity difference of compounds, resulting in different distribution coefficients in different solvents, to achieve the purpose of separation and purification. This method has the characteristics of large injection volume, small solvent consumption, good reproducibility, and high separation efficiency. However, there are no reports on the separation and preparation of non-natural ginsenosides using high-speed countercurrent chromatography. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for separating and preparing non-natural ginsenosides, which solves the problem of separating and preparing effective substances in high-density fermentation products of engineering bacteria.

[0005] The separation and preparation method of the non-natural ginsenosides of the present invention comprises the following steps: using a macroporous adsorption resin to enrich a crude saponin extract I to obtain a crude saponin extract II; and then using high-speed countercurrent chromatography to separate and obtain a saponin extract III, i.e., non-natural ginsenosides. The non-natural ginsenosides are non-natural ginsenosides 3β-O-Glc-DM or non-natural ginsenosides 20S-O-Glc-DM.

[0006] Preferably, the preparation method of the saponin crude extract I is:

[0007] (1-1) Weigh a sample of the fermentation product of engineered yeast Saccharomyces cerevisiae, add an extracting solution, perform reflux extraction for 3 times, each time for 2 hours, combine the filtrates, and concentrate under reduced pressure at 60° C. until there is no alcohol taste, thereby obtaining an extract concentrate; the extracting solution is 70% ethanol by volume, and the mass volume ratio of the sample to the extracting solution is 1 g:10 mL.

[0008] (1-2) The extract was dissolved in distilled water, extracted three times with an equal volume of water-saturated n-butanol, and the n-butanol phases were combined and concentrated under reduced pressure to obtain a crude saponin extract I.

[0009] The saccharomyces cerevisiae engineered bacteria is PgUGT74AE2 saccharomyces cerevisiae engineered bacteria or UGTPg1 saccharomyces cerevisiae engineered bacteria.

[0010] The invention first constructs PgUGT74AE2 brewer's yeast engineering bacteria or UGTPg1 brewer's yeast engineering bacteria for fermentation, and the fermentation product is centrifuged and freeze-dried to obtain the corresponding brewer's yeast engineering bacteria fermentation product sample.

[0011] Preferably, the macroporous adsorption resin is D101 macroporous adsorption resin, and the process for obtaining the crude saponin extract II is: weigh the crude saponin extract I, dissolve it in distilled water and then wet-load it onto the column, and gradiently elute it with 3BV distilled water, 3BV10% ethanol, 3BV30% ethanol, 3BV50% ethanol, 3BV70% ethanol and 3BV90% ethanol respectively, and dry the eluent under reduced pressure to obtain the eluate of the corresponding part, i.e., the crude saponin extract II.

[0012] Preferably, the method of high-speed countercurrent chromatography is: pump the upper phase solution of the solvent system into the separation column at a speed of 20mL / min until the upper phase solution fills the separation column; inject the sample solution into the separation column through the injection loop, adjust the rotation speed, and pump the lower phase solution at a speed of 2mL / min. The detection wavelength is 214nm, and the eluate is collected at intervals of 5min after the peak appears. The eluate is collected for 150min, and the target compound eluate is combined. The white powder after reduced pressure drying is saponin extract III. By LC-MS, 1 H-NMR and 13The structure of saponin extract III was identified by C-NMR, and its purity was determined by standard samples. The purity of non-natural ginsenoside 3β-O-Glc-DM was 96.7%, and the purity of non-natural ginsenoside 20S-O-Glc-DM was 97.5%.

[0013] Preferably, the solvent system is n-hexane:ethyl acetate:methanol:water=1:1:1:1 (volume ratio).

[0014] Preferably, the rotation speed is 800 rpm.

[0015] Studies have found that the ginsenoside 3β-O-Glc-DM prepared by the present invention can inhibit tyrosinase in mouse B16 melanoma cells by 47.9% and melanin synthesis by 45.8%; the ginsenoside 20S-O-Glc-DM can inhibit tyrosinase in mouse B16 melanoma cells by 45.8% and melanin synthesis by 45.3%; therefore, both ginsenosides can be used to prepare drugs for inhibiting melanin production.

[0016] Compared with the prior art, the present invention separates and purifies the fermentation product of brewer's yeast engineered bacteria by high-speed countercurrent chromatography to obtain high-purity non-natural ginsenosides. The method is simple and efficient, and the prepared non-natural ginsenosides show good inhibitory effect on melanin. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the ESI-MS spectrum of compound 3β-O-Glc-DM;

[0018] Figure 2 is the effect of compound 3β-O-Glc-DM on the inhibition rate of tyrosinase synthesis;

[0019] Figure 3 is the effect of compound 3β-O-Glc-DM on the inhibition rate of melanin synthesis;

[0020] Figure 4 is the ESI-MS spectrum of compound 20S-O-Glc-DM;

[0021] Figure 5 is the effect of compound 20S-O-Glc-DM on the inhibition rate of tyrosinase synthesis;

[0022] Figure 6 This is the effect of compound 20S-O-Glc-DM on the inhibition rate of melanin synthesis. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below through specific examples.

[0024] Example 1 Isolation and Preparation of Non-natural Ginsenoside 3β-O-Glc-DM

[0025] The separation and preparation method of non-natural ginsenoside 3β-O-Glc-DM comprises the following specific steps:

[0026] (1) Weigh a sample fermented with PgUGT74AE2 Saccharomyces cerevisiae engineered bacteria, add an extracting solution, perform reflux extraction for 3 times, each time for 2 hours, combine the filtrates, and concentrate under reduced pressure at 60° C. until there is no alcohol taste, thereby obtaining an extract concentrate; the extracting solution is 70% ethanol by volume, and the mass volume ratio of the sample to the extracting solution is 1 g:10 mL;

[0027] (2) The extract was dissolved in distilled water, extracted three times with an equal volume of water-saturated n-butanol, the n-butanol phases were combined, and concentrated under reduced pressure to obtain a crude saponin extract I;

[0028] (3) Weigh the crude saponin extract I, dissolve it in distilled water, and then wet-load it onto a column (D101 macroporous adsorption resin). Gradient elution is performed with 3BV of distilled water, 3BV of 10% ethanol, 3BV of 30% ethanol, 3BV of 50% ethanol, 3BV of 70% ethanol, and 3BV of 90% ethanol, respectively. The 90% ethanol eluate is dried under reduced pressure to obtain the eluate, i.e., the crude saponin extract II;

[0029] (4) The upper phase solution of the solvent system is pumped into the separation column at a rate of 20 mL / min until the upper phase solution fills the separation column; the crude extract II is dissolved in the upper phase solution (sample solution) of the solvent system, and injected into the separation column through the injection loop, the rotation speed is adjusted, and the lower phase solution is pumped into the separation column at a rate of 2 mL / min. The detection wavelength is 214 nm, and the eluate is collected every 5 minutes after the peak appears. The eluate is collected for 150 minutes, and the target compound eluate is combined. The white powder after reduced pressure drying is saponin extract III, i.e., non-natural ginsenoside 3β-O-Glc-DM.

[0030] By LC-MS, 1 H-NMR and 13 The structure of saponin extract III was identified by C-NMR, and its purity was 96.7% as determined by standard samples.

[0031] Example 2 Isolation and Preparation of Non-natural Ginsenoside 20S-O-Glc-DM

[0032] The separation and preparation method of non-natural ginsenoside 20S-O-Glc-DM comprises the following specific steps:

[0033] (1) Weigh a sample of the fermentation product of UGTPg1 Saccharomyces cerevisiae engineered bacteria, add an extracting solution, perform reflux extraction for 3 times, each time for 2 hours, combine the filtrates, and concentrate under reduced pressure at 60° C. until there is no alcohol taste, thereby obtaining an extract concentrate; the extracting solution is 70% ethanol by volume, and the mass volume ratio of the sample to the extracting solution is 1 g:10 mL;

[0034] (2) The extract was dissolved in distilled water, extracted three times with an equal volume of water-saturated n-butanol, the n-butanol phases were combined, and concentrated under reduced pressure to obtain a crude saponin extract I;

[0035] (3) Weigh the crude saponin extract I, dissolve it in distilled water, and then wet-load it onto a column (D101 macroporous adsorption resin). Gradient elution is performed with 3BV of distilled water, 3BV of 10% ethanol, 3BV of 30% ethanol, 3BV of 50% ethanol, 3BV of 70% ethanol, and 3BV of 90% ethanol, respectively. The 70% ethanol eluate is dried under reduced pressure to obtain the eluate, i.e., the crude saponin extract II;

[0036] (4) The upper phase solution of the solvent system is pumped into the separation column at a rate of 20 mL / min until the upper phase solution fills the separation column; the crude extract II is dissolved in the upper phase solution (sample solution) of the solvent system, and injected into the separation column through the injection loop, and the rotation speed is adjusted. At the same time, the lower phase solution is pumped into the separation column at a rate of 2 mL / min, and the detection wavelength is 214 nm. After the peak appears, the eluate is collected every 5 minutes, and the eluate is collected until 150 minutes. The target compound eluate is combined, and the white powder after reduced pressure drying is saponin extract III, i.e., non-natural ginsenoside 20S-O-Glc-DM

[0037] By LC-MS, 1 H-NMR and 13 The structure of saponin extract III was identified by C-NMR, and its purity was 97.5% as determined by standard samples.

[0038] Example 3 Solvent system optimization scheme

[0039] Take the crude extract of saponin I in a 10mL test tube, dissolve it in the following solvent system, take 2mL of the upper and lower phases respectively, blow dry with nitrogen, and dilute to 1mL with methanol. Use HPLC to detect and calculate the distribution coefficient K=A1 / A2, where A1 is the peak area of ​​the upper phase solvent system and A2 is the peak area of ​​the lower phase solvent system. The screening results are shown in Table 1.

[0040] Table 1 Solvent system screening results

[0041]

[0042] Generally speaking, the optimal K value range of high speed countercurrent chromatography is 0.5-2.5, so the screened solvent system is n-hexane: ethyl acetate: methanol: water = 1:1:1:1.

[0043] Example 4 Structural Identification and Analysis of Non-natural Ginsenoside 3β-O-Glc-DM

[0044] According to ESI-MS analysis, [M-H2O] + The ion type m / z is 588.60, and the fragment ion m / z 443.10 is [M-glu] + , the molecular mass of the compound is estimated to be 606.60, such as Figure 1 ; 1 H-NMR (600MHz, DMSO) δ: 1.63(s,3H), 1.56(s,3H), 1.01(s,3H), 0.97(s,3H), 0.91(s,3H), 0.83(s,3H), 0.81(s,3H), 0.75(s,3H); 13 C-NMR (151MHz, DMSO) δ: 88.37 (s, C3), 18.18 (s, C6), 50.36 (s, C17), 77.36 (s, C20) indicated that it contained 8 methyl groups and the core structure was a protopanaxadiol saponin. H :4.85(dd,J=13.0,4.7Hz,1H), δ C : 88.37 (s, C3) indicates that there is a glucose substituent at the C3 position. H :5.10(m,H),δ C : 130.48 (s, C24), 125.71 (s, C25) indicates there is an olefin. C : 77.36 (s), 77.09 (s), 74.43 (s), 73.44 (s), 70.66 (s), indicating that it contained 5 hydroxyl groups, confirming that the compound was 3β-O-Glc-DM.

[0045] Example 5 Structural Identification and Analysis of Non-natural Ginsenoside 20S-O-Glc-DM

[0046] According to ESI-MS analysis, [M+COOH] - The ion type m / z is 651.50, and the estimated molecular mass of the compound is 606.50. Figure 1 ; 1 H-NMR (600MHz, DMSO) δ: 1.63(s,3H), 1.58(s,3H), 1.13(s,3H), 0.93(s,3H), 0.90(s,3H), 0.82(s,3H), 0.79(s,3H), 0.67(s,3H); 13C-NMR (151MHz, DMSO) δ: 81.47 (s, C3), 18.42 (s, C6) indicated that it contained 8 methyl groups and the core structure was a protopanaxadiol saponin. H :5.05(dd,J=16.7,9.7Hz,1H), δ C : 81.47 (s, C3), 97.44 (s, C20) indicating that there is a glucose substituent at the C20 position. H :4.89(m,H),δ C : 130.29 (s, C24), 125.85 (s, C25) indicates an olefin. C : 77.68 (s), 77.24 (s), 76.87 (s), 74.40 (s), 70.71 (s), indicating that it contains 5 hydroxyl groups, confirming that the compound is 20S-O-Glc-DM.

[0047] Example 6 Experiment on the tyrosinase synthesis inhibition rate of non-natural ginsenoside 3β-O-Glc-DM

[0048] When the B16 mouse melanoma cell culture is close to full, it is inoculated on a 96-well plate, 100 μL per well, and 6 replicates are set. Culture in a CO2 incubator for 24 hours, discard the supernatant, add 200 μL of DMEM complete medium with arbutin (positive control group) and 3β-O-Glc-DM at concentrations of 160, 330, 500, 670, and 830 μg / mL, respectively, and continue to culture for 24 hours. After terminating the culture, discard the culture medium, add 1% Triton-100 cell lysis solution to each well, incubate at 37°C for 30 minutes, add 100 μL of 1.0 mg / mL levodopa solution, incubate at 37°C for 30 minutes, and measure the absorbance OD value of each well at a wavelength of 490 nm. Tyrosinase synthesis inhibition rate (%) = (1-OD 实验组 / OD 对照组 )×100%. The results are shown in Figure 2 .

[0049] Example 7 Experiment on the tyrosinase synthesis inhibition rate of non-natural ginsenoside 20S-O-Glc-DM

[0050] 3β-O-Glc-DM was replaced by 20S-O-Glc-DM, and the rest was the same as in Example 6. The results are shown in Figure 5 .

[0051] Example 8 Melanin synthesis inhibition rate experiment of non-natural ginsenoside 3β-O-Glc-DM

[0052] When the B16 mouse melanoma cell culture is close to full, it is inoculated on a 6-well plate, 2 mL per well, and 3 replicates are set. After 24 hours of culture in a CO2 incubator, the supernatant is discarded, and 2 mL of DMEM complete medium with concentrations of 160, 330, 500, 670, and 830 μg / mL of arbutin (positive control group) and 3β-O-Glc-DM are added, and culture is continued for 24 hours. After terminating the culture, the culture medium is discarded, PBS buffer is washed, 1 mL of trypsin is added for digestion, 1 min later, 1640 complete medium is added, digestion is terminated, supernatant is discarded after centrifugation, and 2 mL of PBS buffer is added for washing. 1 mL of 1.0 mol / L NaOH (containing 10% DMSO) is added, and the cells are heated in a water bath at 80°C for 2 hours to break the cells. After terminating the reaction, cool to room temperature, take 100 μL and add it to a 96-well plate, and measure the absorbance OD value of each well at a wavelength of 490 nm. The cells in the control group are cultured normally without any treatment. Melanin synthesis inhibition rate (%) = (1-OD 实验组 / OD 对照组 )×100%. The results are shown in Figure 3 .

[0053] Example 9 Melanin synthesis inhibition rate experiment of non-natural ginsenoside 20S-O-Glc-DM

[0054] 3β-O-Glc-DM was replaced by 20S-O-Glc-DM, and the rest was the same as in Example 8. The results are shown in Figure 6 .

[0055] The above-described embodiments are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Various modifications and alterations to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for separating and preparing non-natural ginsenosides, characterized in that: The saponin crude extract I was enriched by macroporous adsorption resin to obtain saponin crude extract II; then the saponin extract III, i.e., non-natural ginsenosides, was separated by high-speed countercurrent chromatography.

2. The method for separating and preparing non-natural ginsenosides according to claim 1, characterized in that: The non-natural ginsenoside is non-natural ginsenoside 3β-O-Glc-DM or non-natural ginsenoside 20S-O-Glc-DM.

3. The method for separating and preparing non-natural ginsenosides according to claim 1, characterized in that: The process of obtaining saponin crude extract I is as follows: (1-1) Weighing a sample of fermented product of engineered yeast Saccharomyces cerevisiae, adding an extract, performing reflux extraction, combining the filtrate, and concentrating under reduced pressure until there is no alcohol taste, thereby obtaining an extract concentrate; (1-2) The extract was dissolved in distilled water, extracted with an equal volume of water-saturated n-butanol, the n-butanol phases were combined and concentrated under reduced pressure to obtain a crude saponin extract I.

4. The method for separating and preparing non-natural ginsenosides according to claim 3, characterized in that: The saccharomyces cerevisiae engineered bacteria is PgUGT74AE2 saccharomyces cerevisiae engineered bacteria or UGTPg1 saccharomyces cerevisiae engineered bacteria.

5. The method for separating and preparing non-natural ginsenosides according to claim 3, characterized in that: The extracting solution is 70% ethanol by volume, and the mass volume ratio of the sample to the extracting solution is 1 g:10 mL.

6. The method for separating and preparing non-natural ginsenosides according to claim 1, characterized in that: The macroporous adsorption resin is D101 macroporous adsorption resin.

7. The method for separating and preparing non-natural ginsenosides according to claim 1, characterized in that: The process for obtaining saponin crude extract II is as follows: weigh saponin crude extract I, dissolve it in distilled water and then wet-load it onto a column, and gradiently elute it with 3BV distilled water, 3BV10% ethanol, 3BV30% ethanol, 3BV50% ethanol, 3BV70% ethanol and 3BV90% ethanol respectively, and dry the eluent under reduced pressure to obtain the eluate of the corresponding part, namely, saponin crude extract II.

8. The method for separating and preparing non-natural ginsenosides according to claim 1, characterized in that: The specific process of high-speed countercurrent chromatography is as follows: the upper phase solution of the solvent system is pumped into the separation column at a speed of 20 mL / min until the upper phase solution fills the separation column; The sample solution was injected into the separation column through the injection loop, the rotation speed was adjusted, and the lower phase solution was pumped in at a speed of 2 mL / min. The detection wavelength was 214 nm. The eluate was collected every 5 minutes after the peak appeared. The eluate was collected for 150 minutes, and the eluate of the target compound was combined. The white powder after reduced pressure drying was saponin extract III.

9. The method for separating and preparing non-natural ginsenosides according to claim 6, characterized in that: In terms of volume ratio, the solvent system is n-hexane: ethyl acetate: methanol: water = 1:1:1:

1.

10. Use of the non-natural ginsenoside obtained by the method for separating and preparing the non-natural ginsenoside according to any one of claims 1 to 9 in preparing a drug for inhibiting melanin production.

Images