Application of 3-hydroxybutyric acid and 4-pyridoxic acid in synergistically inducing ginseng to accumulate saponins

Abstract

The present invention discloses the application of 3-hydroxybutyric acid and 4-pyridoxic acid to synergistically induce ginseng to accumulate saponin components. The present invention isolates 3-hydroxybutyric acid and 4-pyridoxic acid from the Mesorhizobium sp. G198 strain, and clarifies the effect of the synergistic effect of the two compounds on inducing ginseng to accumulate saponin components. This is the first report of the function of the two compounds inducing ginseng to accumulate saponin components. Based on this, the synergistic application of the two compounds can be used to improve the quality of ginseng.

Description

Technical Field

[0001] The invention belongs to the field of small molecule compounds and relates to the application of 3-hydroxybutyric acid and 4-pyridoxic acid in synergistically inducing ginseng to accumulate saponin components. Background Art

[0002] Ginseng (Panaxginseng CA Meyer) is a common bulk medicinal material. The main medicinal ingredients of ginseng, triterpenoid saponins, have a wide range of pharmacological activities. Ginseng is widely used in clinical applications, mainly for improving people's immunity, preventing cancer and cardiovascular diseases, etc. Long-term use of ginseng can also help to reduce weight and prolong life. The scarcity of high-quality wild ginseng or forest ginseng resources is far from meeting market demand. At present, the ginseng market is mainly dominated by cultivated garden ginseng. However, the cultivated ginseng has a short planting period and low ginsenoside content, resulting in the quality of the mainstream garden ginseng in the market today being far inferior to that of forest ginseng. Therefore, increasing the content of garden ginseng saponins to improve the quality of garden ginseng is of great significance to the healthy development of the cultivated ginseng industry in the future. Summary of the invention

[0003] The purpose of the present invention is to provide a composition for promoting the accumulation of saponin components of ginseng in view of the shortcomings of the prior art.

[0004] Another object of the present invention is to provide application of the composition.

[0005] Another object of the present invention is to provide the use of either or both of 3-hydroxybutyric acid and 4-pyridoxic acid in promoting the accumulation of saponin components in ginseng.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A composition for promoting the accumulation of saponin components in ginseng, comprising 3-hydroxybutyric acid and 4-pyridoxic acid. The concentration of 3-hydroxybutyric acid in the composition is 50 mg / L to 150 mg / L, and the concentration of 4-pyridoxic acid is 50 mg / L to 150 mg / L.

[0008] As a preferred embodiment of the present invention, the concentration of 3-hydroxybutyric acid is 100 mg / L to 150 mg / L, and the concentration of 4-pyridoxic acid is 100 mg / L to 150 mg / L. In some embodiments of the present invention, the concentration of 3-hydroxybutyric acid is 100 mg / L, and the concentration of 4-pyridoxic acid is 100 mg / L; in some embodiments of the present invention, the concentration of 3-hydroxybutyric acid is 100 mg / L, and the concentration of 4-pyridoxic acid is 150 mg / L; in some embodiments of the present invention, the concentration of 3-hydroxybutyric acid is 150 mg / L, and the concentration of 4-pyridoxic acid is 100 mg / L; in some embodiments of the present invention, the concentration of 3-hydroxybutyric acid is 150 mg / L, and the concentration of 4-pyridoxic acid is 150 mg / L.

[0009] The invention discloses an application of the composition in preparing a reagent for promoting the accumulation of saponin components in ginseng.

[0010] As a preferred embodiment of the present invention, the ginsenoside component is any one or more of the ginsenosides of the Rh, F and CK families. The ginsenoside component is selected from one or more of Rg2, Rg3, Rh1, Rh2, Rh4, Rc, CK, F1, F2, F4 and F5.

[0011] As a further preferred embodiment of the present invention, the ginsenoside component is any one or more of Rh and F family ginsenosides.

[0012] In some embodiments of the present invention, the ginsenoside component is selected from Rh2, Rh1, Rg2, Rh4, CK, F2, Rc, F5, F1 and F4 saponins.

[0013] Application of either or both of 3-hydroxybutyric acid and 4-pyridoxic acid in promoting the accumulation of saponin components in ginseng.

[0014] As a preferred embodiment of the present invention, the concentration of 3-hydroxybutyric acid is 100 mg / L to 150 mg / L. In some embodiments of the present invention, the concentration of 3-hydroxybutyric acid is 100 mg / L, 110 mg / L, 120 mg / L, 130 mg / L, 140 mg / L, 150 mg / L; in a preferred embodiment of the present invention, the concentration of 3-hydroxybutyric acid is 150 mg / L.

[0015] As a preferred embodiment of the present invention, the concentration of 4-pyridoxic acid is 100 mg / L to 150 mg / L. In some embodiments of the present invention, the concentration of 4-pyridoxic acid is 100 mg / L, 110 mg / L, 120 mg / L, 130 mg / L, 140 mg / L, 150 mg / L; in a preferred embodiment of the present invention, the concentration of 4-pyridoxic acid is 150 mg / L.

[0016] As a preferred embodiment of the present invention, 3-hydroxybutyric acid and 4-pyridoxic acid are used in combination to promote the accumulation of saponin components in ginseng, wherein the concentration of 3-hydroxybutyric acid is 100mg / L to 150mg / L, and the concentration of 4-pyridoxic acid is 100mg / L to 150mg / L. In some embodiments of the present invention, the concentration of 3-hydroxybutyric acid is 100mg / L, and the concentration of 4-pyridoxic acid is 100mg / L; in some embodiments of the present invention, the concentration of 3-hydroxybutyric acid is 100mg / L, and the concentration of 4-pyridoxic acid is 150mg / L; in some embodiments of the present invention, the concentration of 3-hydroxybutyric acid is 150mg / L, and the concentration of 4-pyridoxic acid is 100mg / L; in some embodiments of the present invention, the concentration of 3-hydroxybutyric acid is 150mg / L, and the concentration of 4-pyridoxic acid is 150mg / L.

[0017] As a preferred embodiment of the present invention, the ginsenoside components are selected from one or more of Rg2, Rg3, Rh1, Rh2, Rh4, Rc, CK, F1, F2, F4, and F5.

[0018] In some embodiments of the present invention, the ginsenoside component is selected from Rh2, Rh1, Rh4, CK, F2, F5, F4 and Rg3 saponins.

[0019] In some embodiments of the present invention, the ginsenoside component is selected from Rh2, Rh1, Rh4, CK, F2, F5 and F1 saponins.

[0020] In other embodiments of the present invention, the ginsenoside components are selected from Rh2, Rh1, Rg2, Rh4, CK, F2, Rc, F5, F1 and F4 saponins.

[0021] Beneficial effects:

[0022] The present invention reports for the first time the activity of two active compounds, 3-hydroxybutyric acid and 4-pyridoxic acid, and their combination in synergistically inducing the accumulation of saponin content, and is expected to be developed into a functional chemical product with the ability to increase the content of ginsenosides and improve the quality of ginseng. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 HPLC analysis results Note: Peaks 1 to 5 represent the representative peaks of A2-2-1 to A2-2-5, respectively, of which Peak 1 is compound A with a retention time of 2.17 min; Peak 5 is compound B with a retention time of 2.92 min.

[0024] Figure 2 The results of the two key compounds (A and B) on the improvement of the total saponin content.

[0025] Figure 3The synergistic effect of the compounds on the improvement of the total saponin content.

[0026] Note: CK represents the blank group without adding compound; synergistic effect represents the combined effect of compound A and compound B. A: compound A (100 mg / L) + compound B (100 mg / L); B: compound A (100 mg / L) + compound B (150 mg / L); C: compound A (150 mg / L) + compound B (100 mg / L); D: compound A (150 mg / L) + compound B (150 mg / L); Different lowercase letters represent significant differences at the 0.05 level.

[0027] Figure 4 Total ion current of mass spectrum of compound A

[0028] Figure 5 Primary mass spectrum of compound A

[0029] Figure 6 Secondary mass spectrum of compound A

[0030] Figure 7 H spectrum of compound A detected by NMR

[0031] Figure 8 Compound A NMR detection C spectrum

[0032] Fig. 9 Compound A structure

[0033] Fig.10 Total ion current of mass spectrum of compound B

[0034] Fig.11 Primary mass spectrum of compound B

[0035] Fig.12 Secondary mass spectrum of compound B

[0036] Fig.13 H spectrum of compound B detected by NMR

[0037] Fig.14 Compound B NMR detection C spectrum

[0038] Fig.15 Compound B structure

[0039] Biomaterial Deposit Information

[0040] G198, classified and named Mesorhizobium sp., is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC No: 30089, and the deposit date is March 21, 2024. DETAILED DESCRIPTION

[0041] Example 1 Preparation of two active compounds: 3-hydroxybutyric acid and 4-pyridoxic acid

[0042] (1) Ginseng adventitious root culture

[0043] The ginseng adventitious root culture liquid system is a 50mL culture bottle, in which the culture medium is 30mL. The formula of the adventitious root culture medium is as follows: 30g sucrose, 5mg IBA hormone (PhytoTech1460, CAS: 133-32-4), 3.35g MS powder (PhytoTech1962, CAS: M519), 1L double distilled water, pH = 6.0. 1g fresh weight of adventitious roots is inoculated, and the liquid culture conditions are 25°C constant temperature dark culture and 120 rpm. When the ginseng adventitious roots grow to 20 days (biomass is 2.5-3.5g fresh weight), G198 (CGMCCNo: 30089) bacteria are ready for inoculation.

[0044] (2) Cultivation of functional strains

[0045] The functional strain G198 (CGMCC No: 30089) was cultured in 1 / 2TSB liquid medium (15g TSB (oxoid, CM0129), 1L distilled water, pH = 6.5, 121℃ high temperature and high pressure sterilization for 25min). G198 was inoculated into the microbial liquid culture medium, and the bacterial solution was obtained after 72h of dark culture at a constant temperature of 28℃ and a speed of 120 rpm. The bacterial solution was centrifuged in a centrifuge (3500 rpm, room temperature) for 15 minutes, and the supernatant was removed to obtain the bacterial precipitate. The precipitate was repeatedly eluted with sterile distilled water and centrifuged twice (same conditions as above) to obtain clean bacterial cells without culture medium components. Finally, the bacterial concentration was fixed to 1*10 by sterile distilled water. 8 CFU / mL concentration for later use. (3) Liquid co-culture of G198 strain and ginseng adventitious roots

[0046] The G198 (CGMCC No: 30089) clean bacterial cell solution prepared above was prepared at a final concentration of 1*10 7 CFU / mL was inoculated into the ginseng adventitious root (20 days) liquid culture system in step (1), and the liquid culture conditions were 25°C constant temperature dark culture, 120 rpm, and co-cultured for 10 days. The obtained co-culture solution was passed through four layers of gauze to obtain a solid particle-free culture solution to be purified and isolated for active products.

[0047] (4) Separation and purification of active products

[0048] Initial separation of aqueous and organic phases

[0049] A total of 200L of co-culture solution was collected and treated with ultrasound (power 500W, frequency 40kHZ) for 30min. The treated co-culture solution was extracted with ethyl acetate (1:1v / v) for 3 times, and the upper ethyl acetate (organic phase) part was collected and the extracts were combined. The organic phase extract was obtained by vacuum distillation (35℃, 0.13MPa). The lower aqueous phase part was distilled under reduced pressure (50℃, 0.13MPa) to obtain the aqueous phase extract part. The aqueous and organic phase extracts were prepared with distilled water and ethyl acetate solvents at a concentration of 10g / L to prepare the mother liquor. Subsequently, the mother liquor was filtered and sterilized with a 0.22μm microporous filter membrane and stored in a refrigerator at 4℃ for use. The above-mentioned 20-day ginseng adventitious root culture system (50mL culture bottle, including 30mL of culture medium) was used to inoculate and co-cultivate according to the final concentration of 1g / L of aqueous and organic phase extracts. The culture conditions were 25°C constant temperature dark culture and 120 rpm. After 10 days of co-culture, the adventitious roots were collected and repeatedly rinsed 3 times with sterile distilled water. The surface moisture of the adventitious roots was then dried with sterile absorbent paper and placed in a 5mL centrifuge tube and dried in a 40°C oven for 24 hours. The dried adventitious roots were collected in a 2ml centrifuge tube, added with zirconium oxide beads, and ground into powder by a tissue crusher (60HZ, 2min) for later detection of saponin content.

[0050] Weigh 30 mg of adventitious root powder accurately into a new 2 ml centrifuge tube, add 1.5 mL of 70% methanol solution (methanol: water = 7:3, v / v), shake and mix thoroughly, let stand for 12 hours, and then ultrasonically extract for 50 minutes (power 500W, frequency 40kHZ) to obtain the extraction solution. Then centrifuge (13000r, room temperature) for 10 minutes to obtain the supernatant, use a 0.22μm microporous filter membrane to remove impurities, and obtain the solution to be tested for saponin content.

[0051] The content of various saponins in adventitious roots was determined by using the UPLC-TQ-MS / MS method. The detection method is as follows: the chromatographic column uses a Waters ACQUITYUPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) column, the column temperature is set to 40°C; the mobile phase is 0.1% formic acid-acetonitrile (A) and 0.1% formic acid-water (B), the flow rate is set to 0.4 mL / min, and the injection volume is 1 μL. The elution gradient was 0-2.5min, 28-31% A; 2.5-3min, 31-35% A; 3-4.5min 35-36% A; 4.5-6min 36-37% A; 6-7min 37-50% A; 7-9min 50-98% A; 9-10min 98-98% A; 10-11min 98-28% A; 11-13min 28% A. Mass spectrometry conditions: In combination with the properties of different saponin compounds, the mass spectrometer was operated in negative ion mode, and the parameters such as Q1 MASS (Da), Q3 MASS (Da), declustering voltage (DP / V), collision energy (CE / eV) and emission voltage (CXP / V) are shown in Table 1 below.

[0052] Table 1 UPLC-TQ-MS / MS mass spectrometry conditions

[0053]

[0054] Note: DP: Declustering potential; CE: Collision energy; CXP: Ejection voltage.

[0055] Combined with the ginsenoside content of different samples, saponin standards were used to determine the saponin quantitative standard curve and its applicable range. The specific standard curve is shown in Table 2.

[0056] Table 2 Regression curve of saponin quantitative calculation

[0057]

[0058]

[0059] Note: The concentration unit is μg / mL.

[0060] Column chromatography purification of the active aqueous fraction

[0061] Based on the active aqueous phase, analytical ODS C 18The column (250×10 mm, 5 μm, Thermo Fisher Scientific, USA) was used for separation. The mobile phase was a methanol-water system. The elution ratio was 10:90, 30:70, 40:60, 60:40, 80:20, 100:0 (v / v). Each gradient had 5 column volumes, and 6 parts were obtained. After the reagents were recovered by vacuum concentration (50°C, 0.13 MPa), 6 samples (Fr.A1 to Fr.A6) were obtained. The adventitious roots were co-cultured one by one at a final concentration of 500 mg / L for each compound to induce the accumulation of saponin content (the induction culture conditions and saponin content detection process were the same as above). The active part (Fr.A2) was identified and then the monomer compound was prepared.

[0062] The A2 fraction was further separated by column chromatography (ODS C 18 The column is the same as above), the mobile phase is methanol-water system, the elution ratio is: 10:90, 20:80, 40:60 (v / v), each gradient is 5 column volumes, and 3 parts are obtained respectively. After the reagent is recovered by vacuum concentration, 3 samples (Fr.A2-1~Fr.A2-3) are obtained, and then the adventitious roots of each section compound are co-cultured at a final concentration of 500 mg / L, and the activity is verified section by section (the induction culture conditions and saponin content detection process are the same as above). It is clear that the active part is in section A2-2.

[0063] Active fraction (A2-2) was separated and purified by HPLC

[0064] The active fraction (Fr.A2-2) was further eluted and separated using high performance liquid chromatography (semi-preparative 3ODS column; YMC, 250×20mm, 5μm). The elution conditions were: column temperature 30°C, flow rate 1mL / min, injection volume 10μL, elution time 30min; elution gradient 15-100% (methanol), where phase A was methanol and phase B was water. Finally, 5 target peaks of monomer compounds were obtained ( Figure 1 As shown), and the monomer compounds were prepared by multiple separations. The monomer chemical components obtained were induced by co-cultivation of adventitious roots at a final concentration of 200 mg / L, and the activity was verified step by step (the induction culture conditions and saponin content detection process were the same as above). Finally, two compounds A2-2-1 (0.187 g, compound A) and A2-2-5 (0.122 g, compound B) were screened and obtained to have significant activity in promoting the accumulation of total saponin content in ginseng.

[0065] (5) Two compounds promote the accumulation of total saponin content in ginseng adventitious roots

[0066] The promotion effect of the two functional ingredients (compounds A and B) was evaluated at concentrations of 50, 100 and 150 mg / L, respectively. A total of 22 saponins were detected in the adventitious root system (as shown in Table 3). The total content of each saponin and the total content of the 22 saponins were determined. The results are as follows Figure 2 As shown in Table 3. Overall, the promoting effect of compound A was greater than that of compound B. The promoting effect increased with the increase of compound concentration. At 100 and 150 mg / L, there were significant differences between the two compounds and the blank group (P≤0.05). The promoting effect was best at a concentration of 150 mg / L. The two compounds increased the total saponin content by 59.2% (compound A) and 54.5% (compound B, Figure 2 and Table 3). After treatment with compound A (150 mg / L), the contents of Rh2, Rh1, Rh4, CK, F2, F5, F4 and Rg3 saponins were significantly higher than those in the control group (P≤0.05, Table 3); after treatment with compound B (150 mg / L), the contents of Rh2, Rh1, Rh4, CK, F2, F5 and F1 saponins were significantly higher than those in the control group (P>0.05, Table 3). Overall, the two compounds stimulated a significant increase in the contents of Rh, F and CK family saponins.

[0067] Table 3 Analysis of the significance of the difference in the content of single saponin

[0068]

[0069] Note: The concentration of compound A and B is 150 mg / L; synergy represents the significant difference of single saponin content after treatment with compound A (150 mg / L) + compound B (150 mg / L). P value less than 0.05 means there is a significant difference at the 0.05 level.

[0070] (6) The two compounds act synergistically to promote the accumulation of total saponin content in ginseng adventitious roots

[0071] The combined application of compound A and compound B has a more prominent promoting effect. Figure 3 As shown in Table 3. When the concentration of compound A (150 mg / L) + compound B (100 mg / L) was used, the total content of saponins increased by 87.5%; when the concentration of compound A (150 mg / L) + compound B (150 mg / L) was used, the total content of saponins increased by 94.6%, and there was no significant difference between the two groups (P>0.05, Figure 3 ). From the changes in the content of a single saponin (Table 3), after the synergistic treatment of compound A (150 mg / L) + compound B (150 mg / L), the content of Rh2, Rh1, Rg2, Rh4, CK, F2, Rc, F5, F1 and F4 saponins was significantly higher than that of the control group (P>0.05). Overall, the compound stimulation mainly increased the content of Rh and F family saponins significantly.

[0072] Example 2 Identification of compound structures by Orbi-Trap-MS / MS and NMR

[0073] The structures of key compounds were identified by using Orbi-Trap-MS / MS. The chromatographic column used was Waters' ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) column, and the column temperature was set at 40°C. The flow rate was set at 0.4 mL / min, the injection volume was 1 μL, and the elution gradient was 0-7 min 2-5% A, 7-12 min 5-10% A, 12-17 min 10-20% A, 17-25 min 20-98% A, where phase A was acetonitrile and phase B was water. ESI was used. - Mode scanning, molecular weight between 50-500Da, bombardment voltage selected 20-60ev.

[0074] Deuterated reagent DMSO (cil) was used as the compound solvent, and the C and H spectra of the compound were detected based on a nuclear magnetic resonance detector (Bruker AVANCE III HD500MHz).

[0075] Compound A, milky white oily liquid, high-resolution electrospray mass spectrometry ESI-MS (negative mode) gave a quasi-molecular ion peak m / z 103.0405 [MH] at 0.67-0.92 min - , the calculated value is 104.0473( Figure 4 and Figure 5 ). In the secondary mass spectrum, m / z103.0406[MH] - is the molecular ion peak, m / z 88.5203 ​​is the fragment ion peak of MH-CH3, and m / z 59.0141 is the fragment ion peak of MH-CO2 ( Figure 6 ).

[0076] The results of NMR data analysis are: 1 H-NMR (DMSO, 600 MHz), δ H :3.974 (1H, m, H-3), 2.261 (2H, m, H-2), 1.090 (3H, d, J=7.2Hz, H-4), such as Figure 7 As shown; 13 C-NMR (DMSO, 150 MHz), δ C :173.314(s,C-1),63.844(d,C-3),44.645(t,C-2),23.889(q,C-4), Figure 8 Based on the above analysis results, the compound was identified as 3-hydroxybutyric acid (C4H8O3), as shown in Fig. 9 shown.

[0077] Compound B, white crystals, high-resolution electrospray mass spectrometry ESI-MS (negative mode) gave a quasi-molecular ion peak m / z 182.0464 [MH] at 2.33-4.49 min - , the calculated value is 183.0532( Fig.10 and Fig.11 ). In the secondary mass spectrum, m / z182.0467[MH] - is the molecular ion peak, m / z 138.0568 is the fragment ion peak of MH-CO2, m / z 110.0616 is the fragment ion peak of MH-CO2-C2H4, m / z 108.0460 is the fragment ion peak of MH-CO2-NO, and m / z 92.0510 is the fragment ion peak of MH-CO2-NO-O ( Fig.12 ).

[0078] The results of NMR data analysis are: 1 H-NMR (DMSO, 600 MHz), δ H : 15.091(1H,s,3-OH), 7.835(1H,s,7-COOH), 4.891(2H,m,H-8), 2.477(3H,s,H-9), 5.547(1H,m,8-OH), such as Fig.13 As shown; 13 C-NMR (DMSO, 150 MHz), δ C :168.490(s,C-7), 162.408(s,C-3), 144.197(s,C-2), 141.504(s,C-6) , 125.173(d,C-5), 123.356(s,C-4), 60.355(t,C-8), 15.227(s,C-9), such as Fig.14 Based on the above results, the compound was identified as 4-pyridoxalic acid (C8H9NO4), as shown in Fig.15 shown.

[0079] Although the two active compounds of 3-hydroxybutyric acid and 4-pyridoxic acid described in the present invention are separated from G198, they have clear structures and are known compounds, so they can be purchased through commercial channels or synthesized by themselves. Both 3-hydroxybutyric acid and 4-pyridoxic acid obtained by the above-mentioned route can be used to solve the technical problems of the present invention and achieve the same or similar technical effects.

Claims

1. A composition for promoting the accumulation of ginsenosides in ginseng, characterized in that: It is composed of 3-hydroxybutyric acid and 4-pyridoxic acid, and the ginsenoside components are any one or more of the ginsenosides of the Rh, F and CK families.

2. Use of the composition according to claim 1 in the preparation of an agent for promoting the accumulation of ginsenoside components of ginseng, wherein the ginsenoside components are any one or more of the ginsenosides of the Rh, F and CK families.

3. The use according to claim 2, characterized in that: The ginsenoside components are selected from one or more of Rg2, Rh1, Rh2, Rh4, Rc, CK, F1, F2, F4, and F5 saponins.

4. The use of any one or both of 3-hydroxybutyric acid and 4-pyridoxalic acid in promoting the accumulation of ginsenoside components in ginseng, wherein the ginsenoside components are any one or more of the ginsenosides of the Rh, F and CK families.

5. The use according to claim 4, characterized in that: The concentration of the 3-hydroxybutyric acid is 100 mg / L to 150 mg / L.

6. The use according to claim 4, characterized in that: The concentration of 4-pyridoxalic acid is 100 mg / L-150 mg / L.

7. The use according to claim 4, characterized in that The invention discloses a method for promoting ginsenoside accumulation in ginseng by combining 3-hydroxybutyric acid and 4-pyridoxic acid, wherein the concentration of the 3-hydroxybutyric acid is 100 mg / L to 150 mg / L, and the concentration of the 4-pyridoxic acid is 100 mg / L to 150 mg / L.

8. The use according to claim 4, characterized in that The ginsenoside components are selected from one or more of Rg2, Rg3, Rh1, Rh2, Rh4, Rc, CK, F1, F2, F4, and F5 saponins.

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