A trollius chinensis extract, a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and specifically discloses a trollius chinensis extract, a preparation method thereof, and application of the trollius chinensis extract in preparation of trollius chinensis granules. The trollius chinensis extract is prepared by using a two-phase low eutectic solvent extraction process. The preparation method can simultaneously extract two types of active ingredients, i.e. hydrophilic and hydrophobic active ingredients, in trollius chinensis, effectively solving the problem of incomplete coverage of ingredients and low extraction yield caused by traditional single solvent extraction. Meanwhile, the two-phase DES used for extraction has the advantages of high extraction efficiency, good biocompatibility, strong adjustability, green environmental protection, and the like, and the extraction process has low energy consumption, high safety, and high industrial feasibility. Compared with the extract prepared by a conventional extraction method, the trollius chinensis extract provided by the application contains both hydrophilic and hydrophobic active ingredients, and the different active ingredients synergistically play a role, so that the antibacterial and anti-inflammatory effect is better. The application can be applied to the preparation of trollius chinensis extract and related oral preparations.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a golden lotus extract, its preparation method, and its application in the preparation of golden lotus granules. Background Technology

[0002] Golden lotus ( Trollius chinensis Bunge Golden lotus (Trollius chinensis) is a perennial herbaceous plant belonging to the Ranunculaceae family. Also known as "dryland lotus," "golden hibiscus," "dryland lotus," or "golden lump," it is widely distributed in Inner Mongolia, Qinghai, Shaanxi, Gansu, Liaoning, Jilin, Henan, Shanxi, and Hebei provinces, with the largest production in Shanxi, Hebei, and Inner Mongolia. Golden lotus has the function of clearing heat and detoxifying, and possesses a wide range of pharmacological effects, including antibacterial, antiviral, antioxidant, antitumor, anti-allergic, and anti-inflammatory analgesic properties. Golden lotus and its preparations are mainly used to treat upper respiratory tract infections, pharyngitis, dental caries, tonsillitis, acute otitis media, allergic rhinitis, and to prevent cardiovascular diseases.

[0003] Golden lotus flowers are rich in various bioactive components, mainly including flavonoids, phenolic acids, alkaloids, and terpenes. Flavonoids are represented by purslane, vitexin, and quercetin, while phenolic acids include veratric acid and vanillic acid. Because golden lotus flowers contain both hydrophilic and hydrophobic bioactive compounds, traditional organic solvent extraction processes are limited by the single polarity of the solvent, typically only efficiently enriching one type of component and failing to simultaneously extract multiple active substances. Extraction processes for hydrophobic components not only suffer from high solvent consumption and energy costs but also pose safety risks due to the flammability and explosiveness of the solvent, and are prone to environmental pollution. Furthermore, their limitations result in a large amount of active ingredients remaining in the raw material matrix, reducing resource utilization and causing resource waste. Therefore, developing a method for the simultaneous and efficient extraction of multiple active components from golden lotus flowers has significant practical value and application significance for promoting the efficient extraction of natural active substances from golden lotus flowers. Summary of the Invention

[0004] In view of the above-mentioned problems existing in the prior art, the present invention provides a golden lotus extract, its preparation method, and its application in the preparation of golden lotus granules. The present invention uses a biphasic eutectic solvent to extract the active ingredients of golden lotus, and the resulting golden lotus extract is enriched with both hydrophilic and hydrophobic active ingredients, resulting in better antibacterial and anti-inflammatory effects. The golden lotus extract obtained by the present invention can be used to prepare various oral dosage forms, including golden lotus granules.

[0005] To achieve the above-mentioned objectives, the embodiments of the present invention employ the following technical solutions: In a first aspect, the present invention provides a method for preparing a golden lotus extract, the method comprising the following steps: S1. Extract the golden lotus flower in a biphasic eutectic solvent with ultrasonic assistance, separate the layers, and obtain a hydrophilic phase extract and a hydrophobic phase extract. S2. The hydrophilic phase extract is purified and eluted through a macroporous adsorption resin column to obtain a hydrophilic phase eluent; S3. The hydrophobic phase extract is purified and eluted using a cation exchange column to obtain a hydrophobic eluent; S4. The hydrophilic eluent and the hydrophobic eluent are mixed and dried to obtain the extract of golden lotus. The biphase eutectic solvent includes hydrophilic eutectic solvents and hydrophobic eutectic solvents; The hydrophilic eutectic solvent includes lactic acid and L-proline; The hydrophobic eutectic solvent includes tetrabutylammonium chloride, lactic acid, menthol, and 1-hexyl-3-methylimidazolium hexafluorophosphate.

[0006] This invention employs a dual-phase eutectic solvent (DES) synergistic extraction process to prepare extracts of *Trollius chinensis*. This method can simultaneously extract both hydrophilic and hydrophobic active ingredients from *Trollius chinensis*, effectively solving the problems of incomplete component coverage and low extraction yield caused by traditional single-solvent extraction. It increases the content of medicinal components such as flavonoids, phenolic acids, alkaloids, and terpenes in the extract. At the same time, the dual-phase DES used in the extraction has advantages such as high extraction efficiency, good biocompatibility, strong adjustability, and green environmental protection. The extraction process has low energy consumption, high safety, and high industrial feasibility.

[0007] This invention achieves efficient extraction of different bioactive components from *Trollius chinensis* by adjusting the type and polarity of hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs) in hydrophilic and hydrophobic DES. This not only avoids the resource waste caused by single-phase DES extraction but also solves the complexity of traditional extraction operations. Furthermore, this invention utilizes a hydrophobic imidazole ionic liquid—1-hexyl-3-methylimidazolium hexafluorophosphate—in a hydrophobic eutectic solvent, which enhances the affinity for hydrophobic pharmacodynamic components and increases their yield.

[0008] Compared to 1-ethyl-3-methylimidazolium hexafluorophosphate, the 1-hexyl-3-methylimidazolium hexafluorophosphate selected in this invention can not only significantly improve the extraction yield of Napier lotus, but also shorten the phase separation time of the two-phase system, avoid emulsification, and improve separation efficiency. At the same time, it can also synergistically form a stable hydrogen bond network and hydrophobic interaction with other components in hydrophobic eutectic solvents such as tetrabutylammonium chloride and menthol, ensuring the stability of the solvent system during the extraction process.

[0009] Preferably, the molar ratio of lactic acid to L-proline is 1.8-2.1:1, and the water content of the hydrophilic eutectic solvent is 14%-20%.

[0010] Preferably, the molar ratio of tetrabutylammonium chloride, lactic acid, menthol and 1-hexyl-3-methylimidazolium hexafluorophosphate is 0.8-1.2:0.8-1.2:0.5-0.8:0.16-0.25, and the water content of the hydrophobic eutectic solvent is 6%-10%.

[0011] In this invention, relying on the presence of ionic liquids (1-hexyl-3-methylimidazolium hexafluorophosphate), an appropriate amount of water is added to the hydrophobic DES to reduce its viscosity and improve extraction efficiency. Adding water not only appropriately increases the fluidity of the hydrophobic DES, thereby enhancing the solvent's permeability to the *Trollius chinensis* cells and the mass transfer efficiency to the target product during extraction, ensuring effective transfer of ultrasonic cavitation energy, and improving the extraction efficiency and yield of the active ingredients; the addition of an appropriate amount of water also helps the hydrophobic components form a more stable hydrogen bond network, ensuring that a clear two-phase interface is quickly and stably formed when subsequently mixed with hydrophilic DES, rather than emulsification, thus improving system stability.

[0012] Preferably, the volume ratio of the hydrophilic eutectic solvent to the hydrophobic eutectic solvent is 1:0.5-1:0.8.

[0013] The specific ratio of hydrophilic eutectic solvent and hydrophobic eutectic solvent in this invention can ensure that the hydrophobic DES is in full contact with the raw material and can cover the surface of the raw material particles, while maintaining the high interfacial tension of the two-phase system, so that clear stratification can be achieved after extraction.

[0014] Preferably, the macroporous adsorption resin column includes an AB-8 resin column or a D101 macroporous adsorption resin column.

[0015] Preferably, the cation chromatography column comprises D001 resin.

[0016] This invention employs a stirring method to prepare a eutectic solvent. HBDs and HBAs are mixed according to a predetermined molar ratio and heated and stirred at 30°C-50°C until a transparent and homogeneous liquid is formed, and no crystals precipitate at room temperature.

[0017] Preferably, the preparation method of the biphase eutectic solvent includes the following steps: first, tetrabutylammonium chloride, lactic acid and menthol are mixed in a preset ratio, heated to 40℃-50℃ and stirred to obtain an initial hydrophobic eutectic solvent; Adding 1-hexyl-3-methylimidazolium hexafluorophosphate to the initial hydrophobic eutectic solvent yields a hydrophobic eutectic solvent. Lactic acid and L-proline are mixed in a predetermined ratio to obtain a hydrophilic eutectic solvent; The hydrophilic eutectic solvent and the hydrophobic eutectic solvent are mixed to obtain the biphase eutectic solvent.

[0018] Compared to using a combination of levulinic acid and proline in the hydrophilic phase, this invention uses lactic acid, which has the same structure in both phases, to avoid "cross-phase contamination," reduce emulsification, entrainment, and loss during extraction, and ensure the stability of the two-phase system. Furthermore, the rational distribution of lactic acid between the hydrophilic and hydrophobic phases can synergistically promote the enrichment of flavonoids, alkaloids, and other components of different polarities in their respective phases, improving the overall extraction rate and component integrity.

[0019] Preferably, the ultrasound-assisted extraction includes the following steps: mixing the golden lotus with a biphasic eutectic solvent at a material-to-liquid ratio of 1:10-1:20, and extracting for 20-40 minutes under ultrasound power of 180W-220W and temperature of 50℃-60℃.

[0020] Preferably, in step S2, an ethanol-water system is used for gradient elution.

[0021] Preferably, in step S3, a methanol-ammonia system is used for gradient elution.

[0022] For example, in step S2, the specific elution conditions are as follows: First stage: Elute with 8%-12% ethanol for 1.5-2.5 BV, flow rate 1.8 BV / h-2.2 BV / h, and collect this eluent. Second stage: elute with 25%-32% ethanol for 1.6 BV-2.2 BV, elute with 48%-52% ethanol for 1 BV-1.5 BV, and collect this effluent; Third stage: Elute with 65%-75% ethanol for 1.6 BV-2.2 BV, and collect this effluent; Combine the eluents to obtain the hydrophilic eluent.

[0023] For example, in step S3, the specific elution conditions are as follows: First stage: 8%-12% methanol + 0.08%-0.13% ammonia solution 1.5BV-1.8BV, collect the solution and discard it; The second stage: A continuous gradient change of the eluent was achieved through a dual-pump mixing system. Within 115-125 minutes, the methanol concentration was increased uniformly from 8%-12% to 35%-45%, and the ammonia concentration was increased uniformly from 0.08%-0.13% to 0.4%-0.6%, maintaining an elution flow rate of 0.8 BV / h-1.0 BV / h throughout. Collection began when the absorbance of the UV detector (280 nm) was ≥0.2 and stopped when the absorbance was ≤0.2, collecting 850 mL of the target eluent. Third stage: 45%-50% methanol + 0.4%-0.6% ammonia solution, elution 0.7 BV-1.2 BV, elution flow rate 0.7 BV / h-1.0 BV / h, absorbance in the collected solution ≤0.2, discard; Post-elution treatment: Wash the resin column with 45%-52% methanol aqueous solution for 1.5 BV-2.5 BV, then wash with deionized water until the pH of the effluent is 5.5-6.2. Collect the second-stage eluent to obtain the hydrophobic eluent.

[0024] In traditional extraction methods, components with different polarities, such as flavonoids, phenolic acids, or alkaloids, often coexist and tend to aggregate due to their polarity differences, forming insoluble particles. The preparation method provided by this invention achieves stepwise enrichment of polar components using a biphasic eutectic solvent: the hydrophilic phase enriches flavonoids and phenolic acids, while the hydrophobic phase enriches hydrophobic components such as alkaloids. These components are then purified using their respective dedicated chromatographic columns and finally mixed as needed. This "separate-then-combine" approach avoids aggregation caused by the coexistence of large amounts of different polar components, allowing the *Trollius chinensis* extract provided by this invention to be rapidly dispersed and dissolved in water.

[0025] Secondly, the present invention provides a golden lotus extract, which is prepared by the method for preparing the golden lotus extract described in the first aspect.

[0026] This invention provides a golden lotus extract rich in hydrophilic active ingredients such as flavonoids (violetin, vitexin, quercetin) and phenolic acids (veratrol, vanillic acid), as well as hydrophobic active ingredients such as alkaloids and terpenes. These different active ingredients work synergistically, resulting in enhanced antibacterial and anti-inflammatory effects. Furthermore, the extract undergoes purification via chromatographic column chromatography, removing impurities that hinder dissolution and improving water solubility. Specifically, purification of the hydrophilic phase extract using a macroporous adsorption resin column effectively removes highly viscous impurities such as polysaccharides, tannins, and water-soluble proteins, preventing them from encapsulating the active ingredients and forming insoluble aggregates. Purification of the hydrophobic phase extract using a cation exchange column enriches alkaloids and other components through ion exchange, while simultaneously removing hydrophobic impurities such as fat-soluble pigments and oils.

[0027] Thirdly, the present invention provides the application of the golden lotus extract provided in the second aspect in the preparation of golden lotus granules.

[0028] The golden lotus granules provided by this invention contain not only golden lotus extract but also pharmaceutically acceptable carriers or excipients. The carriers or excipients include carriers and excipients commonly used in pharmaceuticals, such as at least one of fillers, binders, preservatives, flavoring agents, diluents, or colorants.

[0029] Fourthly, the present invention provides a golden lotus granule, the raw materials for which are made include the golden lotus extract provided in the second aspect. The specific preparation method of the golden lotus granule is as follows: the golden lotus extract is redissolved in ethanol, a filler and a disintegrant are added, and wet granulation is performed to obtain the golden lotus granule.

[0030] Traditional water extraction methods for preparing golden lotus granules have drawbacks such as high impurity content, strong hygroscopicity, poor taste, and large dosage. The golden lotus granules prepared using the golden lotus extract provided by this invention have advantages such as highly concentrated active ingredients, small formulation volume, low dosage, and higher patient compliance. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This is a liquid chromatogram for determining rutin and vitexin in *Trollius chinensis* in this invention. Figure 2 The results of cell viability measurement in the normal group and LPS intervention group in Example 3 of the present invention; Figure 3 The results show the effects of different groups of golden lotus extract on the inflammatory cell model in Example 3 of this invention. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0034] The golden lotus flower raw material selected in this invention is harvested in July in the Mulan Weichang production area of ​​Chengde City, Hebei Province, and is obtained after impurity removal and drying.

[0035] The content of the main active ingredients of the golden lotus used in this invention was determined as follows: total flavonoids (calculated as rutin) 83.6±4.8 mg / g, total alkaloids 0.245±0.06 mg / g, purslane 13.25±1.15 mg / g, vitexin 1.33±0.32 mg / g, quercetin 1.91±0.41 mg / g, veratrum acid 1.17±0.28 mg / g, and vanillic acid 0.85±0.09 mg / g. The liquid chromatograms of purslane and vitexin in the golden lotus are shown below. Figure 1 As shown.

[0036] It should be noted that, before extraction in this invention, the dried golden lotus flower is pulverized and passed through an 80-mesh sieve.

[0037] In this invention, all experiments were repeated 3 times and the average value was taken.

[0038] Example 1 This invention provides a method for preparing a golden lotus extract, the specific preparation method of which is as follows: S1. Construction of a biphasic eutectic solvent and ultrasound-assisted extraction of *Trollius chinensis* (1) Preparation of hydrophilic eutectic solvent Lactic acid and L-proline were mixed at a molar ratio of 2:1 and magnetically stirred at 200 rpm until a homogeneous and transparent liquid was formed. Deionized water was added at 15% of the total mass of lactic acid and L-proline, and the mixture was stirred at 200 rpm at room temperature until the system was homogeneous and transparent, thus obtaining a hydrophilic eutectic solvent. (2) Preparation of hydrophobic eutectic solvent Tetrabutylammonium chloride, lactic acid, and menthol were mixed in a molar ratio of 1:1:0.6, heated to 45°C, and stirred until the system was homogeneous to obtain an initial hydrophobic eutectic solvent. One-third the molar amount of menthol (1-hexyl-3-methylimidazolium hexafluorophosphate) was added to the initial hydrophobic eutectic solvent, and stirring was continued at 45°C until a homogeneous and stable system was formed to obtain an ionic liquid-modified hydrophobic eutectic solvent. 8% by mass of deionized water was added to the ionic liquid-modified hydrophobic eutectic solvent, and stirring was continued at 45°C until the water was completely dispersed and the system formed a homogeneous viscous liquid to obtain the hydrophobic eutectic solvent. (3) Construction and extraction of biphase eutectic solvent The above-mentioned hydrophilic eutectic solvent and hydrophobic eutectic solvent are mixed at a volume ratio of 10:7. After standing, obvious two-phase separation can be observed, thus obtaining the biphase eutectic solvent. Add 500g of *Trollius chinensis* (a type of lotus flower) that has passed through an 80-mesh sieve to 8.5L of the aforementioned biphasic eutectic solvent, transfer the mixture to an ultrasonic extractor, and sonicate at 200W and 55℃ for 30min. The cavitation effect generated by ultrasound effectively disrupts the plant cell wall, promoting the migration of target compounds to the corresponding polar phase: polar components such as flavonoid glycosides preferentially enter the hydrophilic phase, while lipid-soluble terpenes and a small amount of alkaloids enter the hydrophobic phase. After extraction, the mixture was centrifuged at 3000 rpm for 15 min to achieve phase separation, and the lower hydrophilic phase extract and the upper hydrophobic phase extract were collected separately.

[0039] S2. Obtaining the target component from the hydrophilic phase extract Take AB-8 type macroporous adsorption resin, soak it in 95% ethanol for 2 hours to activate it, and then rinse it with deionized water until there is no alcohol odor, ensuring that the pores are open and the surface is clean, to obtain pretreated AB-8 type macroporous adsorption resin; pack the above pretreated AB-8 type macroporous adsorption resin into a column with a specification of φ6.0cm×50cm and a column volume of 1750mL to obtain AB-8 resin column.

[0040] The pH of the hydrophilic extract was adjusted to 3.5 to ensure that the moderately polar organic acids existed in a molecular state, enhancing their hydrophobic interaction with the AB-8 resin and reducing loss during water elution. The sample was loaded onto the AB-8 resin column at a flow rate of 1 BV / h. After loading, the column was eluted with 1.5 BV of deionized water at the same flow rate to remove sugars and inorganic salts. A linear gradient elution was then performed using an ethanol-water system under the following conditions: First stage: Elute with 10% ethanol for 2 BV at a flow rate of 2 BV / h, and collect this eluent. Second stage: 2 BV of elution with 30% ethanol, 1 BV of elution with 50% ethanol, and collect the effluent from this stage. Third stage: Elute 2 BV with 70% ethanol and collect the effluent from this stage; Combine the eluents to obtain the hydrophilic eluent. After elution, rinse the resin column with deionized water at a flow rate of 3 BV / h until neutral. The resin can be recycled.

[0041] S3. Obtaining the target component from the hydrophobic phase extract The D001 resin was packed into a column using a wet packing method with a diameter of φ5.0cm×40cm. The column was regenerated with 3M HCl solution at a flow rate of 1BV / h for 2h, then washed with deionized water until neutral, and finally equilibrated with PBS buffer at pH 6.0. Adjust the pH of the hydrophobic phase extract to 5.5 and load it at a flow rate of 1 BV / h. After the liquid has completely entered the resin column, wash with 2 BV of pH 6.0 PBS buffer to remove unadsorbed impurities, followed by a methanol-ammonia progressive elution (ammonia concentration is volume fraction, based on a methanol-water mixture). The specific elution conditions are as follows: First stage: 10% methanol + 0.1% ammonia solution 1.6 BV, monitored by UV detector (280 nm), stop after the absorbance of the effluent returns to baseline, and discard the collected liquid; The second stage: A continuous gradient change of the eluent was achieved through a dual-pump mixing system. Within 120 minutes, the methanol concentration was increased uniformly from 10% to 40%, and the ammonia concentration was increased uniformly from 0.1% to 0.5%, while maintaining an elution flow rate of 0.9 BV / h throughout the process. Collection began when the absorbance of the UV detector (280 nm) was ≥0.2 and stopped when the absorbance was ≤0.2, yielding 850 mL of the target eluent. Third stage: 50% methanol + 0.5% ammonia solution, elute 1 BV, elution flow rate 0.8 BV / h, for complete desorption, collect liquid with absorbance ≤0.2 and discard; Post-elution treatment: The resin column was rinsed with 50% methanol aqueous solution at 2 BV / h for 2 BV, and then rinsed with deionized water until the pH of the effluent was 6.0.

[0042] Collect the second-stage eluent to obtain a hydrophobic eluent.

[0043] S4. The hydrophilic eluent and the hydrophobic eluent are mixed and concentrated by rotary evaporation under reduced pressure at 45°C and -0.08 MPa. The concentrate is then dried under reduced pressure and pulverized through an 80-mesh sieve to obtain the golden lotus extract.

[0044] Example 2 This invention provides a method for preparing a golden lotus extract, the specific preparation method of which is as follows: S1. Construction of a biphasic eutectic solvent and ultrasound-assisted extraction of *Trollius chinensis* (1) Preparation of hydrophilic eutectic solvent Lactic acid and L-proline were mixed at a molar ratio of 1.8:1 and magnetically stirred at 200 rpm until a homogeneous and transparent liquid was formed. Deionized water was added at 14% of the total mass of lactic acid and L-proline, and the mixture was stirred at 200 rpm at room temperature until the system was homogeneous and transparent, thus obtaining a hydrophilic eutectic solvent. (2) Preparation of hydrophobic eutectic solvent Tetrabutylammonium chloride, lactic acid, and menthol were mixed in a molar ratio of 0.8:0.8:0.5, heated to 40°C, and stirred until the system was homogeneous to obtain an initial hydrophobic eutectic solvent. Half the molar amount of menthol in 1-hexyl-3-methylimidazolium hexafluorophosphate was added to the initial hydrophobic eutectic solvent, and stirring was continued at 40°C until a homogeneous and stable system was formed to obtain an ionic liquid-modified hydrophobic eutectic solvent. 6% by mass of deionized water was added to the ionic liquid-modified hydrophobic eutectic solvent, and stirring was continued at 40°C until the water was completely dispersed and the system formed a homogeneous viscous liquid to obtain the hydrophobic eutectic solvent. (3) Construction and extraction of biphase eutectic solvent The above-mentioned hydrophilic eutectic solvent and hydrophobic eutectic solvent are mixed at a volume ratio of 10:5. After standing, obvious two-phase separation can be observed, thus obtaining the biphase eutectic solvent. Add 500g of golden lotus flowers that have passed through an 80-mesh sieve to 10L of the above-mentioned biphase eutectic solvent, transfer to an ultrasonic extractor, and ultrasonically treat for 40min at 180W and 60℃. After extraction, the mixture was centrifuged at 3000 rpm for 15 min to achieve phase separation, and the lower hydrophilic phase extract and the upper hydrophobic phase extract were collected separately.

[0045] S2. Obtaining the target component from the hydrophilic phase extract AB-8 resin columns were packed according to the method provided in Example 1; Adjust the pH of the hydrophilic phase extract to 3.8, load the sample onto an AB-8 resin column at a flow rate of 1 BV / h, and after loading, rinse with 1 BV of deionized water at the same flow rate to remove sugars and inorganic salts. Then, perform linear gradient elution using an ethanol-water system under the following conditions: First stage: Elute with 10% ethanol at 1.5 BV, flow rate 2 BV / h, and collect this eluent. Second stage: 2 BV of elution with 30% ethanol, 1 BV of elution with 50% ethanol, and collect the effluent from this stage. Third stage: Elute 2 BV with 70% ethanol and collect the effluent from this stage; Combine the eluents to obtain the hydrophilic eluent. After elution, rinse the resin column with deionized water at a flow rate of 3 BV / h until neutral. The resin can be recycled.

[0046] S3. Obtaining the target component from the hydrophobic phase extract A hydrophobic eluent was prepared according to the method described in Example 1.

[0047] S4. The hydrophilic eluent and the hydrophobic eluent are mixed and concentrated by rotary evaporation under reduced pressure at 45°C and -0.06 MPa. The concentrate is then dried under reduced pressure and pulverized through an 80-mesh sieve to obtain the extract of golden lotus.

[0048] Example 3 This invention provides a method for preparing a golden lotus extract, the specific preparation method of which is as follows: S1. Construction of a biphasic eutectic solvent and ultrasound-assisted extraction of *Trollius chinensis* (1) Preparation of hydrophilic eutectic solvent Lactic acid and L-proline were mixed at a molar ratio of 2.1:1 and magnetically stirred at 200 rpm until a homogeneous and transparent liquid was formed. Deionized water was added at 20% of the total mass of lactic acid and L-proline, and the mixture was stirred at 200 rpm at room temperature until the system was homogeneous and transparent, thus obtaining a hydrophilic eutectic solvent. (2) Preparation of hydrophobic eutectic solvent Tetrabutylammonium chloride, lactic acid, and menthol were mixed in a molar ratio of 1.2:1.2:0.8, heated to 50°C, and stirred until the system was homogeneous to obtain an initial hydrophobic eutectic solvent. One-quarter of the molar amount of menthol in 1-hexyl-3-methylimidazolium hexafluorophosphate was added to the initial hydrophobic eutectic solvent, and stirring was continued at 50°C until a homogeneous and stable system was formed to obtain an ionic liquid-modified hydrophobic eutectic solvent. 10% by mass of deionized water was added to the ionic liquid-modified hydrophobic eutectic solvent, and stirring was continued at 50°C until the water was completely dispersed and the system formed a homogeneous viscous liquid to obtain the hydrophobic eutectic solvent. (3) Construction and extraction of biphase eutectic solvent The above-mentioned hydrophilic eutectic solvent and hydrophobic eutectic solvent are mixed at a volume ratio of 10:8. After standing, obvious two-phase separation can be observed, thus obtaining the biphase eutectic solvent. Add 500g of golden lotus flowers that have passed through an 80-mesh sieve to 9.0L of the above-mentioned biphase eutectic solvent, transfer to an ultrasonic extractor, and ultrasonically treat for 20min at 220W and 50℃. After extraction, the mixture was centrifuged at 3000 rpm for 15 min to achieve phase separation, and the lower hydrophilic phase extract and the upper hydrophobic phase extract were collected separately.

[0049] S2. Obtaining the target component from the hydrophilic phase extract Take D101 macroporous adsorption resin, soak it in 95% ethanol for 4 hours to activate it, and then rinse it with deionized water until the effluent has no alcohol odor, ensuring that the pores are open and the surface is clean, to obtain pretreated D101 macroporous adsorption resin; pack the above pretreated D101 macroporous adsorption resin into a column with a size of φ6.0cm×50cm and a column volume of 1800mL to obtain a D101 resin column.

[0050] Elute and collect the eluent according to the method provided in Example 1 to obtain a hydrophilic eluent.

[0051] S3. Obtaining the target component from the hydrophobic phase extract A hydrophobic eluent was prepared according to the method described in Example 1.

[0052] S4. The hydrophilic eluent and the hydrophobic eluent are mixed and concentrated by rotary evaporation under reduced pressure at 45°C and -0.09 MPa. The concentrate is then dried under reduced pressure and pulverized through an 80-mesh sieve to obtain the golden lotus extract.

[0053] Example 4 This invention provides a method for preparing golden lotus granules, which includes the following steps: Take 36.5g of the golden lotus extract prepared in Example 1, add 65mL of 70% ethanol solution, place on a magnetic stirrer, stir at 200rpm at room temperature until the extract is completely dissolved to obtain a golden lotus extract complex solution. 103.5g of dextrin (passed through an 80-mesh sieve) and 69.0g of sucrose (passed through a 100-mesh sieve) were placed together in a three-dimensional mixer. The mixing speed was adjusted to 150 rpm, and the mixture was mixed for 30 minutes. The mixture was then transferred to a gyratory granulator, and the above-mentioned golden lotus extract complex solution was slowly sprayed at 100 rpm while stirring until a soft material that could be formed "clumped together by hand but crumbled easily when touched" was formed. After the soft material was prepared, the granulator was started, a 10-mesh sieve was selected, and granulation was performed. The wet granules that passed through the sieve were collected, spread evenly on a sterile tray, and placed in a forced-air drying oven. The oven was dried at 60℃ and a wind speed of 2 m / s for 3 hours, during which the granules were turned over every 30 minutes to ensure uniform drying. The dried granules were obtained.

[0054] The dried granules are then fed back into a gyratory granulator, with the upper sieve screen at 10 mesh and the lower sieve screen at 80 mesh, to granulate and obtain uniform golden lotus granules; each 1g of golden lotus granules contains 2.4g of raw golden lotus medicinal material.

[0055] Comparative Example 1 The present invention provides a comparative example of a method for preparing a golden lotus extract, the specific preparation method of which is as follows: Take 500g of golden lotus flowers that have passed through an 80-mesh sieve, add 6L of purified water, soak overnight, heat and reflux for 1 hour (based on boiling time), and then filter while hot through a 300-mesh sieve to obtain water extract I and residue I. Add 5L of purified water to the dregs I, heat and reflux for 1 hour (based on boiling time), then filter while hot through a 300-mesh sieve to obtain water extract II and dregs II. Combine water extract I and water extract II, and concentrate under reduced pressure at 55℃ and -0.08MPa until the relative density of the thick paste reaches 1.40 (measured at 50℃). Then stop the concentration, dry under reduced pressure, and pulverize through an 80-mesh sieve to obtain the golden lotus extract.

[0056] Comparative Example 2 The present invention provides a comparative example of a method for preparing a golden lotus extract, which is basically the same as that in Example 1, except that “1-hexyl-3-methylimidazolium hexafluorophosphate” in step S1 is replaced with an equal amount of “1-ethyl-3-methylimidazolium hexafluorophosphate”. The other raw materials and their amounts, operating steps and process parameters are not changed, and the golden lotus extract is finally obtained.

[0057] Comparative Example 3 The present invention provides a comparative example of a method for preparing a golden lotus extract, which is basically the same as that in Example 1. The only difference is that "lactic acid" in "preparation of hydrophilic eutectic solvent in step S1 (1)" is replaced with an equal amount of "acetylpropionic acid". The other raw materials and their amounts, operating steps and process parameters are not changed, and the golden lotus extract is finally obtained.

[0058] Example 1 The weight statistics of the golden lotus extracts prepared in different comparative examples and embodiments of the present invention are shown in Table 1.

[0059] Table 1

[0060] Note: Different lowercase letters indicate significant differences (P < 0.05).

[0061] As shown in Table 1, the dry extract yield of the Golden Lotus extract prepared by the conventional water extraction method in Comparative Example 1 of the present invention was 29.8%, which was significantly higher than that of the Golden Lotus extract prepared by the extraction and purification of the extract by the biphasic eutectic solvent in Examples 1-3 and Comparative Examples 2-3 of the present invention. However, the antibacterial and anti-inflammatory effects of the extracts obtained by different preparation methods need to be verified by subsequent experiments.

[0062] Regarding the methods for extracting and purifying *Trollius chinensis* extract using biphasic eutectic solvents, the extract yields of the methods provided in Examples 1-3 of this invention are higher than those in Comparative Examples 2-3. Specifically, the extract yield of the method provided in Example 1 is significantly higher than that of Comparative Examples 2-3.

[0063] Example 2 This invention compares the antibacterial activity of extracts from *Staphylococcus aureus* and *Escherichia coli* prepared by different methods, using *Trollius chinensis* as examples. The specific details are as follows: (1) Activation and propagation of strains Escherichia coli and Staphylococcus aureus were removed from the -80℃ freezer and placed in a clean bench. Using a sterile inoculation loop, the frozen bacterial suspensions were streaked onto nutrient agar plates for separation. Single colonies of well-formed E. coli or Staphylococcus aureus were selected and inoculated into sterile nutrient broth medium, and incubated at 37℃ with shaking at 200 rpm for 12 hours. The bacterial suspensions were then diluted to a 0.5 McFarland concentration using NB medium.

[0064] (2) Preparation of the medicine solution Accurately weigh the extracts of golden lotus prepared in Examples 1-3 or Comparative Examples 1-3, add a certain volume of 20% DMSO solution, and sonicate for 5 minutes to fully dissolve them, so as to prepare a drug solution with a concentration of 50 mg / mL.

[0065] (3) Measurement of MIC and MBC The minimum inhibitory concentration (MIC) was determined using the two-fold dilution method. Specifically, 100 μL of liquid culture medium was added to each well of a 96-well plate. After adding an equal volume of drug solution to the first well, a concentration gradient was prepared using a serial dilution method (wells 1-10). Each well was thoroughly mixed during the dilution process, and 100 μL of the mixture was discarded from the 10th well.

[0066] Set up a control group: In well 11, add 100 μL of sterile saline as a negative control; in well 12, add 25 μg / mL of penicillin as a positive control.

[0067] The treated 96-well plates were incubated in a 37°C incubator for 18 hours, and the results were observed.

[0068] The minimum bactericidal concentration (MBC) was determined using the plate spread method. Specifically, 10 μL of culture was inoculated into an antibiotic-free agar plate from the wells in the MIC experiment that showed no bacterial growth. After incubation at 37°C for 18 h, the lowest drug concentration in which no bacteria grew in three replicate experiments was taken as the MBC value.

[0069] The results of the determination of MIC and MBC of different test bacteria by extracts of golden lotus prepared by different methods are shown in Table 2.

[0070] Table 2

[0071] Note: - indicates no antibacterial effect at the maximum concentration.

[0072] As shown in Table 2, the extracts of *Trollius chinensis* provided in Examples 1-3 and Comparative Examples 1-3 of this invention all have a certain inhibitory effect on *Escherichia coli* and *Staphylococcus aureus*. Among them, the extracts of *Trollius chinensis* provided in Examples 1-3 and Comparative Examples 2-3 have better antibacterial effects against both pathogenic bacteria.

[0073] Example 3 This invention investigates the restorative effects of different extracts from *Trollius chinensis* on an inflammation model constructed using RAW264.7 cells. A dedicated culture medium for RAW264.7 cells was used for culturing the cells. Details are as follows: (1) CCK-8 assay for cell viability After different drug treatments, the culture medium in each group of original 96-well plates was discarded, and 100 μL of culture medium and 10 μL of CCK-8 were added to each well. After incubation in an incubator (37℃, 5% CO2) for 2 hours, the absorbance was measured at 450 nm using an ELISA reader.

[0074] (2) Establishment of an inflammatory cell model Low concentrations of lipopolysaccharide (LPS) can induce cellular inflammatory responses, promote the secretion of inflammatory factors, and cause cell damage by activating the PI3K / Akt / mTOR signaling pathway. Therefore, this invention uses LPS-induced inflammation in RAW264.7 cells to construct an inflammation model. Specifically, the steps include: selecting RAW264.7 cells in good growth condition in RAW264.7-specific culture medium, and seeding them into 96-well plates, with each well containing 10... 5 100 μL of cell culture with CFU / mL was added and cultured overnight. After cell adhesion, RAW264.7 cells in the model group were treated with 12 μg / mL LPS for 24 h (37℃, 5% CO2). The normal group was not treated with LPS. Each group was repeated in 3 replicates.

[0075] The cell viability measurements of the normal group and the LPS intervention group are as follows: Figure 2 As shown.

[0076] Depend on Figure 2 It can be seen that LPS at a concentration of 12 μg / mL has a significant inhibitory effect on cell proliferation, with a cell survival rate of only 80.5%, indicating that an inflammatory cell model was successfully constructed after intervention with this concentration of LPS.

[0077] (3) Intervention of Golden Lotus Extract on Inflammatory Cell Models The final concentration of *Trollius chinensis* extract provided in Examples 1-3 and Comparative Examples 2-3 was added to the wells of different inflammation models to a final concentration of 50 μg / mL. Based on the relationship between the weight of the *Trollius chinensis* extract provided in Example 1 (approximately 13.7 g of raw medicinal material per 1 g of extract) and the weight of the *Trollius chinensis* extract provided in Comparative Example 1 (approximately 3.35 g of raw medicinal material per 1 g of extract), the final concentration of the *Trollius chinensis* extract provided in Comparative Example 1 was 204 μg / mL. After adding the *Trollius chinensis* extracts of different groups, the cell viability of the different groups was measured after 6 hours of intervention in the inflammation cell models; the inflammation cell model group was not intervened and was cultured for only 6 hours, serving as the model group.

[0078] The effects of different groups of nasturtium extract on inflammatory cell models are as follows: Figure 3 As shown.

[0079] Depend on Figure 3 It is evident that the *Trollius chinensis* extracts provided in Examples 1-3 and Comparative Examples 1-3 of this invention can reduce cellular inflammatory responses. With equal amounts of raw medicinal materials, the anti-inflammatory effect of the *Trollius chinensis* extract provided in Example 1 is significantly superior to the *Trollius chinensis* extract prepared by conventional water extraction in Comparative Example 1.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a golden lotus extract, characterized in that: The preparation method includes the following steps: S1. Extract the golden lotus flower in a biphasic eutectic solvent with ultrasonic assistance, separate the layers, and obtain a hydrophilic phase extract and a hydrophobic phase extract. S2. The hydrophilic phase extract is purified and eluted through a macroporous adsorption resin column to obtain a hydrophilic phase eluent; S3. The hydrophobic phase extract is purified and eluted using a cation exchange column to obtain a hydrophobic eluent; S4. The hydrophilic eluent and the hydrophobic eluent are mixed and dried to obtain the extract of golden lotus. The biphase eutectic solvent includes hydrophilic eutectic solvents and hydrophobic eutectic solvents; The hydrophilic eutectic solvent includes lactic acid and L-proline; The hydrophobic eutectic solvent includes tetrabutylammonium chloride, lactic acid, menthol, and 1-hexyl-3-methylimidazolium hexafluorophosphate.

2. The method for preparing the golden lotus extract as described in claim 1, characterized in that: The molar ratio of lactic acid to L-proline is 1.8-2.1:1; and / or The molar ratio of the tetrabutylammonium chloride, lactic acid, menthol, and 1-hexyl-3-methylimidazolium hexafluorophosphate is 0.8-1.2:0.8-1.2:0.5-0.8:0.16-0.25; and / or The hydrophilic eutectic solvent has a water content of 14%-20%; and / or The hydrophobic eutectic solvent has a water content of 6%-10%; and / or The volume ratio of the hydrophilic eutectic solvent to the hydrophobic eutectic solvent is 1:0.5-1:0.

8.

3. The method for preparing the golden lotus extract as described in claim 1, characterized in that: The macroporous adsorption resin column includes an AB-8 resin column or a D101 macroporous adsorption resin column; and / or The cation chromatography column includes D001 resin.

4. The method for preparing the golden lotus extract as described in claim 1 or 2, characterized in that: The preparation method of the dual-phase eutectic solvent includes the following steps: Tetrabutylammonium chloride, lactic acid and menthol are mixed in a predetermined ratio and heated to 40℃-50℃ with stirring to obtain an initial hydrophobic eutectic solvent. Adding 1-hexyl-3-methylimidazolium hexafluorophosphate to the initial hydrophobic eutectic solvent yields a hydrophobic eutectic solvent. Lactic acid and L-proline are mixed in a predetermined ratio to obtain a hydrophilic eutectic solvent; The hydrophilic eutectic solvent and the hydrophobic eutectic solvent are mixed to obtain the biphase eutectic solvent.

5. The method for preparing the golden lotus extract as described in claim 1, characterized in that: The ultrasound-assisted extraction includes the following steps: mixing the golden lotus with a biphasic eutectic solvent at a material-to-liquid ratio of 1:10-1:20, and extracting for 20-40 minutes at an ultrasonic power of 180W-220W and an extraction temperature of 50℃-60℃.

6. The method for preparing the golden lotus extract as described in claim 1, characterized in that: In step S2, gradient elution is performed using an ethanol-water system; and / or In step S3, a gradient elution is performed using a methanol-ammonia system.

7. A golden lotus extract, characterized in that: It is prepared by the method of any one of claims 1-6 for the preparation of the golden lotus extract.

8. The use of the golden lotus extract according to claim 7 in the preparation of golden lotus granules.

9. A type of golden lotus granule, characterized in that: The raw materials used to make it include the golden lotus extract as described in claim 7.

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