Method for rapidly preparing high-purity phlorizin and trilobatin from lithocarpus litseifolius and application thereof

Abstract

The present application belongs to the technical field of natural plant active ingredient separation and purification, and provides a method for rapidly preparing high-purity phloridzin and trilobatin from Lithocarpus phyllochlamys and application thereof. Leaf blades of Lithocarpus phyllochlamys are added with an organic solvent for extraction, residue is removed by filtration, and the filtrate is concentrated under reduced pressure to obtain an extraction extract; the extraction extract is added into hot distilled water for stirring and dissolving, supernatant is separated by standing, and the precipitate is collected; the precipitate is added into an alcohol-water mixed solvent, and after dissolution, activated carbon is added for decolorization, and filtration is performed to obtain a refined liquid; the refined liquid is separated by preparative high-performance liquid chromatography, a methanol-water mixed solution is used as a mobile phase, trilobatin and phloridzin characteristic elution components are collected respectively, and after each component is concentrated under reduced pressure and vacuum dried, pure phloridzin and trilobatin products with a purity of ≥98% are obtained. The process route of the present application is simple, relies on directional extraction with an organic solvent, water washing for impurity removal, and activated carbon decolorization combined pretreatment, greatly simplifies the process, and the solvent can be recycled and reused, has low loss, and is green and environmentally friendly.

Description

Technical Field

[0001] This invention belongs to the field of natural plant active ingredient separation and purification technology, specifically involving a method and application for the rapid preparation of high-purity phlorizin and trifolin from Litsea cubeba. More specifically, it describes a process for simultaneously preparing phlorizin and trifolin monomers with a purity ≥98% using dried sweet Litsea cubeba tea leaves as raw materials, and the industrial application of the obtained high-purity products in the food, cosmetics, and pharmaceutical fields. Background Technology

[0002] Litsea cubeba, commonly known as sweet tea, is a unique medicinal and edible tree species in southern my country. Its leaves are rich in two types of dihydrochalcone active substances: phlorizin and trifolin. Phlorizin has pharmacological effects such as lowering blood sugar, protecting the liver, and anti-inflammation, while trifolin has outstanding advantages in anti-oxidation, skin whitening, antibacterial preservation, and its demand in the fields of health food, pharmaceutical raw materials, and cosmetic additives has been increasing year by year.

[0003] Existing processes for preparing active ingredients from Litsea cubeba leaves mostly employ pure water reflux extraction combined with alcohol precipitation, resin chromatography, or low-temperature crystallization. These processes are lengthy, have poor selectivity for impurity removal, and struggle to completely remove large amounts of pigments, tannins, and water-soluble polysaccharides from the raw materials. Most processes can only prepare one product at a time, making simultaneous purification of both components difficult and resulting in significant fluctuations in the purity of the finished product, which fails to meet the quality requirements of high-end pharmaceutical raw materials and standard reference materials. Therefore, developing a novel process combining directional organic solvent extraction with simplified activated carbon decolorization and one-step liquid chromatography separation of the two components has significant industrialization value. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method and application for the rapid preparation of high-purity phlorizin and trifolin from Litsea cubeba, aiming to solve the problems mentioned in the background art.

[0005] In a first aspect, the present invention provides a method for rapidly preparing high-purity phlorizin and trifolin from Litsea cubeba, comprising the following steps: Step 1: Add organic solvent to extract the leaves of Litsea cubeba, filter to remove residue, concentrate the filtrate under reduced pressure to obtain the extract. Step 2: Add the extracted extract to hot distilled water, stir to dissolve, let stand to separate the supernatant, and collect the precipitate; that is, discard the supernatant rich in polysaccharides, proteins and water-soluble ash, and retain the precipitate enriched with phlorizin and trifolin. Step 3: Add the precipitate to the alcohol-water mixed solvent, dissolve it, add activated carbon for decolorization, filter to remove activated carbon, adsorbed pigments and fat-soluble impurities, and obtain the refined liquid. Step 4: The refined liquid is separated by preparative high performance liquid chromatography. The mobile phase is a methanol-water mixture. The characteristic eluents of trifolin and phlorizin are collected separately. After concentration under reduced pressure and vacuum drying, trifolin and phlorizin are obtained.

[0006] Furthermore, in step one, the leaves of *Litsea cubeba* are either fresh or dried.

[0007] Furthermore, in step one, the organic solvent is selected from any one of ethanol, methanol, acetone, n-butanol, and dichloromethane.

[0008] Furthermore, in step one, the extraction is a reflux extraction, the ratio of the Litsea cubeba leaf to the organic solvent is 1g:8-25mL, the extraction temperature is 65-85℃, the extraction is performed 2-3 times, and the extraction time for each extraction is 40-90min.

[0009] Furthermore, in step two, the ambient temperature for standing is 4–8°C, and the standing time is 18–24 hours.

[0010] Furthermore, in step three, the alcohol-water mixed solvent is an aqueous methanol solution; the amount of activated carbon added is 0.5-3.5% of the precipitate mass; the decolorization temperature is 40-60℃; and the stirring decolorization time is 20-60 min.

[0011] Furthermore, in step four, the volume ratio of the methanol-water mixture is methanol:water = 32:68 to 48:52.

[0012] Furthermore, in step four, the preparation of high performance liquid chromatography uses a C18 reversed-phase preparative chromatographic column, with a flow rate of 7–14 mL / min, a detection wavelength of 280 nm, and a column temperature of 23–30 °C.

[0013] Furthermore, in step four, the vacuum drying temperature is 42–58°C, and the vacuum degree is -0.082–-0.096 MPa.

[0014] Secondly, the present invention provides an application of high-purity phlorizin and trifolin obtained rapidly from Litsea cubeba, wherein the high-purity phlorizin and trifolin are used to prepare biopharmaceutical preparations, functional health foods, skin care cosmetics or natural food antioxidant and preservative additives.

[0015] The present invention has the following technical effects: Phlorizin and trifolin, prepared by this process with a purity ≥98%, can be applied in three major fields: ① Pharmaceutical field: preparation of raw materials and pharmaceutical intermediates for hypoglycemic, anti-inflammatory, and liver and kidney protection; ② Health food field: development of dietary supplements for adjuvant hypoglycemia and antioxidant effects, and functional health foods; ③ Daily chemical and food preservation: preparation of raw materials for anti-aging, whitening, and skin care, and natural food antioxidant and preservative additives.

[0016] Compared with existing technologies, this invention employs directional extraction with organic solvents to selectively enrich dihydrochalcone target compounds. A large number of water-soluble macromolecular impurities are directly removed in the subsequent washing step, eliminating the need for traditional macroporous resin packing and gradient elution processes, thus shortening the production cycle. Activated carbon provides low-cost, one-time decolorization, efficiently removing various colored impurities and trace lipids. The pretreatment steps are simple and the production cost is low. Preparative liquid chromatography accurately separates trifolin and phlorizin components, simultaneously obtaining monomers with over 98% high purity. The product's structure is verified by NMR spectroscopy to be consistent with the standard, allowing it to be used directly as a chemical reference. The extraction solvent can be distilled, recovered, and recycled, resulting in low overall organic solvent loss. The process is environmentally friendly and can be industrialized for continuous production. Attached Figure Description

[0017] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures: Figure 1 The HPLC chromatogram for the purity determination of phlorizin prepared in an embodiment of the present invention is shown below. Figure 2 The HPLC purity chromatogram of trifolin prepared in an embodiment of the present invention; Figure 3 The ¹³C-NMR nuclear magnetic resonance spectrum of phlorizin prepared for the example; Figure 4 The ¹H-NMR nuclear magnetic resonance spectrum of phlorizin prepared for the example; Figure 5 The ¹³C-NMR nuclear magnetic resonance spectrum of trifolin prepared for the example; Figure 6 The ¹H-NMR nuclear magnetic resonance spectrum of trifolin prepared for the example. Detailed Implementation

[0018] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0020] Example 1: Rapid preparation of high-purity phlorizin and trifolin from Litsea cubeba. Fresh leaves of *Litsea cubeba* (100g dry weight) were added to anhydrous ethanol at a ratio of 1:8g / mL and mixed thoroughly. The extraction temperature was controlled at 65℃, and the mixture was refluxed twice under sealed conditions, with each extraction lasting 40 minutes. After extraction, the plant solids were removed by filtration, and the filtrates were combined and concentrated to obtain the extract. Hot distilled water was added, and the mixture was stirred thoroughly and allowed to stand at 4℃ for 18 hours. The supernatant containing water-soluble polysaccharides and proteins was discarded, and the precipitate was collected. The precipitate was dissolved thoroughly in a 10% (v / v) methanol aqueous solution, and activated carbon was added at 0.5% of the precipitate mass. The decolorization temperature was controlled at 40℃, and the mixture was stirred at this temperature for 20 minutes. After decolorization, the mixture was filtered while hot to remove the activated carbon and adsorbed pigments and lipid-soluble impurities, yielding a clear and purified liquid. The liquid was filtered through a microporous membrane and injected into a C18 reversed-phase preparative column for separation. The chromatographic conditions were: mobile phase methanol-water (v / v) 32:68, flow rate 7 mL / min, detection wavelength 280 nm, and column temperature 23℃. Based on the chromatographic peak patterns, the characteristic eluents of trifolin and phlorizin were collected separately; the eluents of the two components were concentrated under reduced pressure and then dried to constant weight under vacuum.

[0021] Chromatogram for HPLC purity determination of phlorizin is shown below. Figure 1 As shown; the chromatogram for HPLC purity determination of trifolin is as follows. Figure 2 As shown; the ¹³C-NMR spectrum of phlorizin is as follows Figure 3 As shown; the ¹H-NMR nuclear magnetic resonance spectrum of phlorizin is as follows. Figure 4 As shown; the ¹³C-NMR spectrum of trifolin is as follows. Figure 5 As shown; the ¹H-NMR nuclear magnetic resonance spectrum of trifolin is as follows. Figure 6 As shown in the figure. The results showed that, as determined by HPLC external standard method, the purity of trifolin was 98.12% and the purity of phlorizin was 98.05%, both with a purity ≥98%; NMR spectroscopy confirmed that the product structure was consistent with the standard.

[0022] Specific data for NMR structure identification: The pure phlorizin and trifolin obtained in Example 1 of this invention were subjected to nuclear magnetic resonance detection, with deuterated methanol as the solvent. The specific spectral data are as follows: 1. Phlorizin NMR data 1) Simultaneously, phenolic rings (7.04 / 6.69), phloroglucinol A ring (6.09), and β-glucose terminal hydrogen (4.59, J=7.8 Hz) are observed, consistent with the characteristics of phlorizin (containing glucose); the multiplets in the sugar region of 3.30–4.83 ppm are the hydrogen signals of the six-membered pyranose ring, which superimpose with the dihydrochalcone aglycone signal to form the phlorizin hydrogen spectral fingerprint.

[0023] 2) The carbon spectral data were in perfect agreement with the standard phlorizin NMR data, proving that the product is a glucose-substituted phlorizin structure, and the structural identification was accurate.

[0024] 2. Trilobatin NMR data 1) The hydrogen signal mentioned above is completely consistent with the characteristic hydrogen proton signal of trifolin, which is consistent with the structural characteristics of the trifolin dihydrochalcone skeleton.

[0025] 2) The carbon signal showed no interference from impurity peaks and was consistent with the published carbon spectrum data of trifolin, confirming that the product was trifolin.

[0026] Example 2: Rapid preparation of high-purity phlorizin and trifolin from Litsea cubeba Fresh leaves of *Litsea cubeba* (100g dry weight) were added to methanol at a ratio of 1:15 g / mL and mixed thoroughly. The extraction temperature was controlled at 72℃, and the mixture was extracted twice under sealed reflux, with each extraction lasting 60 min. After extraction, the plant solids were removed by filtration, and the filtrates were combined and concentrated to obtain the extract. Hot distilled water was added, and the mixture was stirred thoroughly and allowed to stand at 6℃ for 24 h. The supernatant containing water-soluble polysaccharides and proteins was discarded, and the precipitate was collected. A 10% (v / v) methanol aqueous solution was added to dissolve the precipitate completely, and activated carbon was added at 2% of the precipitate mass. The decolorization temperature was controlled at 50℃, and the mixture was stirred at this temperature for 35 min. After decolorization, the mixture was filtered while hot to remove the activated carbon and adsorbed pigments and lipid-soluble impurities, yielding a clear and purified liquid. The liquid was filtered through a microporous membrane and injected into a C18 reversed-phase preparative column for separation. The chromatographic conditions were: mobile phase methanol-water (v / v) 40:60, flow rate 10 mL / min, detection wavelength 280 nm, and column temperature 25℃. Based on the chromatographic peak patterns, the characteristic eluents of trifolin and phlorizin were collected separately; the eluents of the two components were concentrated under reduced pressure and then dried to constant weight under vacuum.

[0027] HPLC analysis showed that the purity of trifolin was 99.15% and that of phlorizin was 98.82%, meeting the requirement of ≥98% purity. The structure was confirmed by NMR.

[0028] Example 3: Rapid preparation of high-purity phlorizin and trifolin from Litsea cubeba Fresh Litsea cubeba leaves (100g dry weight) were added to acetone at a material-to-liquid ratio of 1:25g / mL and mixed thoroughly. The extraction temperature was controlled at 85℃, and the mixture was refluxed three times under sealed conditions, with each extraction lasting 90 minutes. After extraction, the plant solids were removed by filtration, and the filtrates were combined and concentrated to obtain the extract. Hot distilled water was added, and the mixture was stirred thoroughly and allowed to stand at 8℃ for 24 hours. The supernatant containing water-soluble polysaccharides and proteins was discarded, and the precipitate was collected. The precipitate was dissolved thoroughly in a 10% (v / v) methanol aqueous solution, and activated carbon was added at 3.5% of the precipitate mass. The decolorization temperature was controlled at 60℃, and the mixture was stirred at this temperature for 60 minutes. After decolorization, the mixture was filtered while hot to remove the activated carbon and adsorbed pigments and fat-soluble impurities, yielding a clear and purified liquid. The liquid was filtered through a microporous membrane and injected into a C18 reversed-phase preparative column for separation. The chromatographic conditions were: mobile phase methanol-water (v / v) 48:52, flow rate 14 mL / min, detection wavelength 280 nm, and column temperature 30℃. Based on the chromatographic peak patterns, the characteristic eluents of trifolin and phlorizin were collected separately; the eluents of the two components were concentrated under reduced pressure and then dried to constant weight under vacuum.

[0029] HPLC external standard method analysis showed that the purity of trifolin was 98.26% and that of phlorizin was 98.11%, both exceeding 98% purity; the structure was confirmed by NMR.

[0030] Comparative Example 1: 100g of dried Litsea cubeba leaves were added to distilled water at a material-to-liquid ratio of 1:20 g / mL and mixed thoroughly. The extraction temperature was controlled at 95℃, and the mixture was extracted twice under sealed reflux, with each extraction lasting 60 min. After extraction, the plant solid residue was removed by filtration, and the filtrates were combined and concentrated to obtain an extract. 95% ethanol was added to adjust the alcohol concentration to 70%, and the mixture was allowed to stand at 6℃ for 18 h. Macromolecular impurities were removed by filtration, and the ethanol was recovered to obtain an alcohol-free extract. The extract was directly fed into a preparative liquid chromatography system without water washing or activated carbon decolorization, and the liquid chromatography conditions were the same as in Example 2. The collected components were dried to obtain a powder.

[0031] Test results: The purity of the product trifolin was 86.37%, and the purity of phlorizin was 84.92%. There were many impurity peaks and serious pigment residue, which could not meet the 98% high purity standard. The sample was dark yellow and showed oxidative discoloration after 7 days of storage.

[0032] Application Example 1: Oral hypoglycemic tablets The high-purity phlorizin prepared according to this invention is used as the core active ingredient, combined with conventional pharmaceutical excipients such as starch, microcrystalline cellulose, magnesium stearate, and sodium carboxymethyl starch. Phlorizin is mixed evenly with fillers and disintegrants according to the formula ratio, and an appropriate amount of binder is added to form a soft mass. After granulation and sizing, a lubricant is added and thoroughly mixed. The mixture is then tableted using a tableting machine to produce oral hypoglycemic tablets. These tablets, with phlorizin as the main active substance, can lower blood sugar and protect the liver and kidneys. They are suitable for daily management of high blood sugar in individuals, and the product is stable and convenient to take.

[0033] Application Example 2: Natural Sweeteners in Food High-purity trifolin obtained through this process is selected as a natural sweetener. Trifolin has high sweetness and pure flavor, while also possessing antioxidant and health benefits, and can replace some sucrose and artificial sweeteners. By adding trifolin to beverages, pastries, preserved fruits, dairy products, and other food systems according to the formula ratio, and mixing, blending, sterilizing, and filling / forming with conventional food ingredients, excellent-flavored sweet foods can be produced. This sweetener is derived from natural plants, has high safety, and has no side effects like artificial sweeteners. It can also impart certain antioxidant properties to food, broadening the development direction of health foods.

[0034] 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, and 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 rapidly preparing high-purity phlorizin and trifolin from Litsea cubeba, characterized in that, Includes the following steps: Step 1: Add organic solvent to extract the leaves of Litsea cubeba, filter to remove residue, concentrate the filtrate under reduced pressure to obtain the extract. Step 2: Add the extracted extract to hot distilled water, stir to dissolve, let stand to separate the supernatant, and collect the precipitate; Step 3: Add the precipitate to the alcohol-water mixed solvent, dissolve it, add activated carbon for decolorization, filter, and obtain the purified liquid. Step 4: The refined liquid is separated by preparative high performance liquid chromatography. The mobile phase is a methanol-water mixture. The characteristic eluents of trifolin and phlorizin are collected separately. After concentration under reduced pressure and vacuum drying, trifolin and phlorizin are obtained.

2. The method according to claim 1, characterized in that: In step one, the leaves of Litsea cubeba are either fresh or dried.

3. The method according to claim 1, characterized in that: In step one, the organic solvent is selected from any one of ethanol, methanol, acetone, n-butanol, and dichloromethane.

4. The method according to claim 1, characterized in that: In step one, the extraction is a reflux extraction, the ratio of the Litsea cubeba leaf and the organic solvent is 1g:8-25mL, the extraction temperature is 65-85℃, the extraction is performed 2-3 times, and the extraction time for each extraction is 40-90min.

5. The method according to claim 1, characterized in that: In step two, the ambient temperature for standing is 4–8°C, and the standing time is 18–24 hours.

6. The method according to claim 1, characterized in that: In step three, the alcohol-water mixed solvent is an aqueous methanol solution; the amount of activated carbon added is 0.5-3.5% of the precipitate mass; the decolorization temperature is 40-60℃; and the stirring decolorization time is 20-60 min.

7. The method according to claim 1, characterized in that: In step four, the volume ratio of the methanol-water mixture is methanol:water = 32:68 to 48:

52.

8. The method according to claim 1, characterized in that: In step four, the preparation of high performance liquid chromatography uses a C18 reversed-phase preparative column, with a flow rate of 7–14 mL / min, a detection wavelength of 280 nm, and a column temperature of 23–30 °C.

9. The method according to claim 1, characterized in that: In step four, the vacuum drying temperature is 42–58°C, and the vacuum degree is -0.082–-0.096 MPa.

10. The application of high-purity phlorizin and trifolin prepared by the method according to any one of claims 1 to 9, characterized in that: The high-purity phlorizin and trifolin shown are used to prepare biopharmaceutical preparations, functional health foods, skin care cosmetics, or natural food antioxidant and preservative additives.

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