Preparation method and application of gleditsia sinensis saponin-silver ion antibacterial chelate
The preparation of saponin-silver ion chelates by eutectic solvent extraction and ultrasound-assisted chelation reaction solves the problem of weak antibacterial activity of saponin and enables its widespread application in cosmetics and daily chemical products.
Patent Information
- Application Number
- CN202410939568.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-15
- Publication Date
- 2026-01-16
AI Technical Summary
The antibacterial activity of saponins is relatively weak, which limits their widespread application in functional surfactants, and no research has been reported on silver ion-enhanced saponins.
Saponins were extracted using a eutectic solvent and purified by macroporous resin. Subsequently, they were chelated with silver nitrate under light-protected conditions, and ultrasonic assisted reaction was used to prepare saponins-silver ion chelates.
It significantly enhances the antibacterial activity of saponins and improves their silver ion solubility under alkaline conditions, making them a highly efficient, green, and environmentally friendly antibacterial agent, thus broadening their application in cosmetics and daily chemical products.
Smart Images
Figure FT_1 
Figure FT_2 
Figure FT_3
Abstract
Description
Technical Field
[0001] This invention relates to the field of natural product deep processing technology, and in particular to a method for preparing and applying a saponin-silver ion antibacterial chelate. Background Technology
[0002] *Gleditsia sinensis* Lam., also known as soapberry, is a perennial tree or small tree belonging to the Caesalpinioideae subfamily of the Fabaceae family. *Gleditsia sinensis* possesses excellent properties such as cold resistance, drought resistance, nitrogen fixation, water conservation, and resistance to diseases and pests, making it an important tree species for ecological restoration. It is distributed throughout both northern and southern my country, with relatively abundant resources. In recent years, the processing and utilization of *Gleditsia sinensis* resources has become a research hotspot. Studies have found that the fruit shell of *Gleditsia sinensis* contains a large amount of pentacyclic triterpenoid saponins, with a content of about 20-30%, making it the woody plant resource with the highest known saponin content. *Gleditsia sinensis* saponins are amphiphilic and are natural nonionic surfactants, showing good application prospects in daily chemical products.
[0003] However, the bioactivity of saponins from saponins is weak, especially their antibacterial activity, which is inferior to that of other plant-derived saponins (such as tea saponins and quinoa saponins). With consumers increasingly demanding green functional surfactants in cosmetics and daily chemical products, the weak antibacterial ability of saponins from saponins greatly limits their widespread application as functional surfactants. Therefore, enhancing the antibacterial activity of saponins from saponins and achieving their widespread application is urgently needed. Silver ions have good antibacterial ability and are often used as highly effective antibacterial agents. However, research on enhancing the antibacterial activity of saponins from saponins using silver ions has not yet been reported. Saponin molecules have two sugar chains containing a large number of active hydroxyl groups, which are expected to coordinate with silver ions to form stable chelates. This would not only improve the antibacterial activity of saponins from saponins but also solve the problem of silver ion solubility being easily affected by pH and salt type. It would have a green, environmentally friendly, safe, and highly effective antibacterial effect. Summary of the Invention
[0004] The purpose of this invention is to provide a method for preparing and applying a saponin-silver ion antibacterial chelate, which enables saponin to have green, safe, broad-spectrum and highly efficient antibacterial activity, thereby broadening the application field of saponin as a green functional surfactant.
[0005] To achieve the above objectives, the present invention adopts the following technical solution:
[0006] (1) Saponin was extracted using a eutectic solvent. After dialysis, the extract was purified using a macroporous resin and dried to obtain high-purity saponin.
[0007] (2) Using the prepared saponin as raw material, it is fully dissolved in distilled water and silver nitrate is added to carry out a chelation reaction. After the chelation is completed, it is dialyzed, the dialysate is centrifuged and the supernatant is collected. After freeze-drying, saponin-silver ion chelate is obtained.
[0008] The eutectic solvent mentioned in step (1) is a clear and transparent liquid synthesized with choline chloride as hydrogen bond acceptor (HBA) and lactic acid as hydrogen bond donor (HBD). The molar ratio of HBA to HBD is 1:2, and the water content of the eutectic solvent is 10-30%.
[0009] The extraction process described in step (1) uses an ultrasonic-assisted low-eutectic solvent to extract saponins, with a material-to-liquid ratio of 1:10-30, an ultrasonic power of 200-600W, and an ultrasonic time of 3-20min.
[0010] The molecular weight cutoff of the dialysis bag described in step (1) is 500-1000 Da.
[0011] The macroporous resin type mentioned in step (1) is either AB-8 or D101.
[0012] In step (1), after the macroporous resin is purified and the sample is loaded for adsorption, it is first eluted with distilled water for 2-4 column volumes, and then eluted with ethanol solution with a volume fraction of 80-95%. The eluent rich in saponins is collected, concentrated under reduced pressure, and lyophilized.
[0013] The chelation reaction described in step (2) is carried out under light-protected conditions, with a chelation reaction temperature of 30-60℃, a pH of 4-7, and a saponin / silver nitrate mass ratio of 4:1-14:1.
[0014] The chelation reaction described in step (2) requires first dissolving saponin in distilled water at a mass fraction of 0.1-1%, adjusting the pH with 0.1M NaOH and HNO3, and then adding silver nitrate to carry out the chelation reaction.
[0015] The chelation reaction described in step (2) is accompanied by ultrasound assistance, with an ultrasound power of 200-500W and an ultrasound time of 2-10 minutes.
[0016] The dialysis bag described in step (2) has a molecular weight cutoff range of 500-1000 Da, the dialysate (outside the bag) is distilled water, and the dialysis time is 48-72 h.
[0017] Beneficial effects
[0018] (1) This invention uses forest processing residues as raw materials, which are inexpensive and widely available.
[0019] (2) The method of using a low eutectic solvent combined with ultrasound assistance can greatly improve the efficiency of obtaining saponins from saponins. At the same time, by using the specific molar ratio of choline chloride-lactic acid low eutectic solvent and water selected by this invention, the target saponins can be selectively obtained.
[0020] (3) The use of ultrasound-assisted chelation reaction can enhance the collision frequency between saponin molecules and silver ions, improve the ability of saponin to chelate silver ions, significantly improve product quality, greatly enhance antibacterial activity, and significantly improve preparation efficiency compared with traditional stirring chelation.
[0021] (4) This invention can solve the problems of poor antibacterial ability and low added value of soap pod saponin, and provide the possibility for the development and application of soap pod saponin in high-value cosmetics and daily chemical products. It is of great significance to improve the utilization rate and economic value of soap pod resources. Attached Figure Description
[0022] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings:
[0023] Figure 1 The infrared spectrum of the saponin-silver ion chelate is shown.
[0024] Figure 2 This is a surface elemental distribution diagram of the saponin-silver ion chelate from saponins;
[0025] Figure 3 The foaming ability of the saponin-silver ion chelate. Detailed Implementation
[0026] The following embodiments are examples of the present invention and should not be regarded as limiting the present invention.
[0027] Example 1: Screening of low eutectic solvents for saponins from saponins
[0028] In this embodiment, defatted soapberry pod powder was used as raw material. The soapberry pod powder was extracted using a low eutectic solvent. The types of low eutectic solvents were screened, and the results are shown in Table 1.
[0029] Table 1. Effects of different eutectic solvent types on the extraction efficiency of saponins from saponins.
[0030]
[0031] Example 2: Extraction of saponins from saponins using a low-melting solvent
[0032] Saponins were extracted from saponins using a preferred choline chloride-lactic acid eutectic solvent (molar ratio 1:2) under ultrasonic assistance. The effects of various factors on the extraction efficiency of saponins were investigated, including solvent moisture content (10, 20, 30, 40, 50%), liquid-to-solid ratio (20:1, 30:1, 40:1, 50:1, 60:1 mL / g), ultrasonic power (200, 300, 400, 500, 600 W), and extraction time (2, 8, 11, 14, 17 min). The results are shown in Table 2. The results indicate that under the preferred conditions, the extraction efficiency of saponins is greater than 180 mg / g.
[0033] Table 2. Effects of different extraction conditions on the extraction efficiency of saponins from saponins.
[0034]
[0035] Example 3: Purification of Saponins from Gleditsia sinensis
[0036] The extract from Example 2 was further dialyzed using a 500-1000 Da dialysis bag. The dialysate in the bag was purified using AB-8 or D101 macroporous resin. After loading and adsorption, the sample was eluted with distilled water for 2-4 column volumes, and then eluted with 80-95% ethanol solution. The saponin-rich eluent was collected, concentrated under reduced pressure, and lyophilized. The obtained saponin had a purity greater than 50%.
[0037] Example 4: Preparation of Saponin-Silver Ion Antibacterial Chelate
[0038] Using the saponin extracted and purified in Example 3 as raw material, it was fully dissolved in distilled water. The pH of the saponin solution was adjusted to 4-7 using 0.1M NaOH and HNO3. Silver nitrate was added at a saponin / silver ion mass ratio of 4:1-14:1. The temperature was adjusted to 30-60℃, and an ultrasonic chelation reaction was carried out under light-protected conditions. The ultrasonic power was 200-500W, and the ultrasonic time was 2-15 minutes. After chelation, the solution was dialyzed with distilled water for 48-72 hours using a dialysis bag with a molecular weight cutoff of 500-1000 Da. After freeze-drying, a saponin-silver ion antibacterial chelate was obtained, with a silver ion content greater than 8 mg / g.
[0039] Example 5: Determination of Silver Ion Solubility
[0040] The saponin-silver ion antibacterial chelate was dissolved in distilled water of different pH values, and the supernatant was collected after centrifugation. After wet digestion of the supernatant, the solubility of silver ions was determined using inductively coupled plasma atomic emission spectrometry (ICP-AES). The solubility of silver ions in the chelate was greater than 85% under alkaline conditions (pH 9.0).
[0041] Example 6: Determination of the antibacterial activity of saponin-silver ion chelate
[0042] The minimum inhibitory concentration (MIC) of the saponin-silver ion chelate against *Escherichia coli* and *Staphylococcus aureus* was determined using the two-fold isocratic dilution method. Both strains were purchased from the China Industrial Microbial Culture Collection Center. The strains were inoculated into liquid culture medium and cultured at 37°C with shaking until the logarithmic growth phase. The cultured solution in the logarithmic growth phase was then diluted to 1×10⁻⁶ using LB liquid medium. 5 CFU / mL ready for use. Add 100 μL of saponin-silver ion chelate solution to each well of a 96-well plate, then add an equal volume of the above bacterial suspension and mix thoroughly. Incubate the 96-well plate at 37°C with gentle shaking for 24 hours, and then measure the OD. 600 The MICs of saponin-silver ion chelates against Escherichia coli and Staphylococcus aureus were determined.
[0043] Example 7 used defatted soapberry pod powder as raw material. The extraction solvent was choline chloride-lactic acid (molar ratio 1:2), the water content in the eutectic solvent was 30%, the liquid-to-solid ratio was 30 mL / g, the ultrasonic power was 400 W, the extraction time was 14 min, and the extraction efficiency of soapberry saponin was 180 mg / g. The extract was treated with a dialysis bag with a molecular weight cutoff of 500 Da for 72 h, and the solution in the bag was purified using AB-8 macroporous resin. After eluting with distilled water for 3 column volumes, elution was performed with 90% ethanol solution. The saponin-rich eluent fraction was collected, concentrated under reduced pressure, and lyophilized to obtain soapberry saponin with a purity of 67.3%.
[0044] The saponin obtained above was fully dissolved in distilled water. The pH of the saponin solution was adjusted to 6.0 using 0.1M NaOH and HNO3. Silver nitrate was added at a saponin / silver nitrate mass ratio of 10:1. The chelation temperature was adjusted to 40℃, and the solution was ultrasonically treated for 8 min in the dark at a power of 300W, followed by stirring for 15 min. After chelation, the solution was treated with a dialysis bag with a molecular weight cutoff of 500 Da for 72 h. The solution in the bag was then freeze-dried to obtain the saponin-silver ion chelate. The silver ion solubility of the obtained chelate at pH 9.0 was 91.3%, and the silver ion content in the saponin was 8.06 mg / g. The MICs of the saponin-silver ion chelate against Escherichia coli and Staphylococcus aureus are shown in Table 3.
[0045] Table 3. MIC (mg / mL) of saponins and their silver ion chelates against Escherichia coli and Staphylococcus aureus.
[0046]
[0047] Example 8 used defatted soapberry pod powder as raw material. The extraction solvent was choline chloride-lactic acid (molar ratio 1:2), the water content in the eutectic solvent was 40%, the liquid-to-solid ratio was 30 mL / g, the ultrasonic power was 400 W, the extraction time was 17 min, and the extraction efficiency of soapberry saponin was 182 mg / g. The extract was treated with a dialysis bag with a molecular weight cutoff of 500 Da for 72 h, and the solution in the bag was purified using AB-8 macroporous resin. After eluting with distilled water for 2 column volumes, elution was performed with 90% ethanol solution. The saponin-rich eluent fraction was collected, concentrated under reduced pressure, and lyophilized to obtain soapberry saponin with a purity of 70.1%.
[0048] The saponin obtained above was fully dissolved in distilled water. The pH of the saponin solution was adjusted to 7.0 using 0.1M NaOH and HNO3. Silver nitrate was added at a saponin / silver nitrate mass ratio of 12:1. The chelation temperature was adjusted to 50℃, and the solution was ultrasonically treated for 10 min in the dark at a power of 400W, followed by stirring for 20 min. After chelation, the solution was treated with a dialysis bag with a molecular weight cutoff of 500 Da for 72 h. The solution in the bag was then freeze-dried to obtain the saponin-silver ion chelate. The silver ion solubility of the obtained chelate at pH 9.0 was 87.4%, and the silver ion content in the saponin was 8.21 mg / g. The MICs of the saponin-silver ion chelate against Escherichia coli and Staphylococcus aureus are shown in Table 4.
[0049] Table 4. MIC (mg / mL) of saponins and their silver ion chelates against Escherichia coli and Staphylococcus aureus.
[0050]
[0051] The method for preparing the saponin-silver ion chelate provided in this invention is simple and easy to implement. Saponin has a strong ability to chelate silver ions, and the resulting chelate is stable, exhibits high solubility under alkaline conditions, and shows significantly improved inhibitory activity against *Escherichia coli* and *Staphylococcus aureus* compared to saponin alone. It is a green, environmentally friendly, safe, and highly effective antibacterial agent. The saponin-silver ion chelate prepared by this invention holds promise for use as a green antibacterial agent in functional foods, daily chemical products, and other fields. Therefore, this invention is of great significance for the refined utilization of *Gleditsia sinensis*.
[0052] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.
Claims
1. A preparation method and application of a Gleditsia sinensis soap-silver ion bacteriostatic chelate, characterized by the following steps: (1) Gleditsia sinensis soap is extracted using a eutectic solvent, the extract is dialyzed, and then purified using a macroporous resin to obtain high-purity Gleditsia sinensis soap after drying; (2) The Gleditsia sinensis soap obtained is used as a raw material, dissolved in distilled water, and then silver nitrate is added for ultrasonic chelation. After chelation, dialysis is performed, the supernatant is obtained by centrifugation of the dialysate, and then freeze-drying is performed to obtain a Gleditsia sinensis soap-silver ion bacteriostatic chelate. The chelate can effectively inhibit Escherichia coli and Staphylococcus aureus.
2. The preparation method and application of the Gleditsia sinensis soap substance-silver ion bacteriostatic chelate according to claim 1, characterized in that: The eutectic solvent in step (1) is a clear and transparent liquid synthesized using choline chloride as a hydrogen bond acceptor (HBA) and lactic acid as a hydrogen bond donor (HBD). The molar ratio of HBA to HBD is 1:2, and the water content of the eutectic solvent is 10-30%.
3. The preparation method and application of the Gleditsia sinensis sugar- silver ion bacteriostatic chelate according to claim 1, characterized in that: In step (1), the extraction process uses ultrasonic-assisted eutectic solvent to extract soap. The solid-liquid ratio is 1:10-30, the ultrasonic power is 200-600 W, and the ultrasonic time is 3-20 min.
4. The preparation method and application of the Gleditsia sinensis soap substance-silver ion bacteriostatic chelate according to claim 1, characterized in that: The dialysis bag in step (1) has a molecular weight cutoff range of 500-1000 Da.
5. The preparation method and application of the Gleditsia sinensis soap substance-silver ion bacteriostatic chelate according to claim 1, characterized in that: The macroporous resin in step (1) is one of AB-8 or D101.
6. The preparation method and application of the Gleditsia sinensis soap substance-silver ion bacteriostatic chelate according to claim 1, characterized in that: In step (1), the macroporous resin is purified. After adsorption, distilled water is used to elute 2-4 column volumes, and then an ethanol solution with a volume fraction of 80-95% is used for elution. The elution fraction rich in soap is collected, concentrated under reduced pressure, and freeze-dried.
7. The preparation method and application of the Gleditsia sinensis soap substance-silver ion bacteriostatic chelate according to claim 1, characterized in that: The chelation reaction in step (2) is carried out in the dark. The chelation reaction temperature is 30-60 ℃, the pH is 4-7, and the mass ratio of soap to silver ion is 4:1-14:
1.
8. The preparation method and application of the Gleditsia sinensis soap substance-silver ion bacteriostatic chelate according to claim 1, characterized in that: In step (2), the chelation reaction requires that the soap be dissolved in distilled water with a mass fraction of 0.1-1%. After adjusting the pH using 0.1 M NaOH and HNO3, silver nitrate is added for chelation.
9. The preparation method and application of the Gleditsia sinensis soap substance-silver ion bacteriostatic chelate according to claim 1, characterized in that: The chelation reaction in step (2) is assisted by ultrasonic, with an ultrasonic power of 200-500 W and an ultrasonic time of 2-15 minutes.
10. The preparation method and application of the Gleditsia sinensis soap substance-silver ion bacteriostatic chelate according to claim 1, characterized in that: The dialysis bag in step (2) has a molecular weight cutoff range of 500-1000 Da, and the dialysate (outside the bag) is distilled water. The dialysis time is 48-72 h.
11. The preparation method and application of the Gleditsia sinensis soap substance-silver ion bacteriostatic chelate according to claim 1, characterized in that: The Gleditsia sinensis soap-silver ion chelate has a silver ion solubility of more than 85% at pH 9.
0.
12. The preparation method and application of the Gleditsia sinensis soap substance-silver ion bacteriostatic chelate according to claim 1, characterized in that: The Gleditsia sinensis soap-silver ion chelate can be used as a bacteriostatic active factor, a functional surfactant, and a bacteriostatic additive to prevent and treat infectious diseases caused by Escherichia coli and Staphylococcus aureus.